Single-domain VH H antibodies directed to norovirus GI.1 and GII.4 and their use

ABSTRACT

Isolated VHH monoclonal antibodies are disclosed that specifically bind to a Norovirus polypeptide. In some embodiments, the Norovirus is a Genogroup I Norovirus or a Genogroup II Norovirus. In other embodiments, the Norovirus is Norwalk or MD2004 virus. In some embodiments, the monoclonal antibodies specifically bind VP1. Also disclosed are compositions including the disclosed antibodies, nucleic acids encoding these antibodies, expression vectors including the nucleic acids, and isolated host cells that express the nucleic acids. The antibodies and compositions disclosed herein can be used for detecting the presence of a Norovirus in a biological sample, or detecting a Norovirus infection. Also disclosed are methods of treating and/or preventing a NoV infection.

CROSS REFERENCE TO RELATED APPLICATION(S)

This is a continuation of U.S. patent Ser. No. 15/982,998, filed on May17, 2018, which is a continuation of U.S. patent application Ser. No.14/889,774, filed on Nov. 6, 2015, issued as U.S. Pat. No. 10,000,556,which is the U.S. National Stage of International Application No.PCT/US2014/037520, filed May 9, 2014, which was published in Englishunder PCT Article 21(2), which claims the benefit of U.S. ProvisionalApplication No. 61/821,354, filed May 9, 2013. The prior applicationsare incorporated by reference herein their entirety.

FIELD OF THE DISCLOSURE

This relates to the field of antibodies, specifically to single-domainantibodies that specifically bind a Norovirus polypeptide, such as aNorwalk or MD2004 virus polypeptide, and their use.

BACKGROUND

Norovirus (NoV) is a non-enveloped virus that belongs to the familyCaliciviridae. It constitutes the mayor cause of epidemicgastroenteritis in close settings (Green, Caliciviridae: TheNoroviruses. Fields Virology, 6^(th) edition. H. P. Knipe, et al. (eds),published by Philadelphia: Lippincott Williams & Wilkins: 582-608,(ISBN-13: 978-1451105636), 2013) and since the introduction of rotavirusvaccines, norovirus has become the leading cause of medically attendedacute gastroenteritis in U.S. children, associated with nearly 1 millionhealth care visits annually (Payne, et al., N Engl J Med 368(12):1121-30, 2013). A gastroenteritis episode due to NoV is incapacitatingduring the acute phase that usually lasts from 1 to 3 days and includesexplosive vomiting, stomach cramps and diarrhea Immunocompetent patientsusually recover completely from the illness but the gastroenteritis maybe severe in young children, the elderly and immunocompromised,increasing the risk for morbidity and mortality (Kaufman et al.,Antiviral Res 105C: 80-91, 2014). It was estimated that around 200,000children die annually because of NoV gastroenteritis, especially indeveloping countries (Patel et al., Emerg Infect Dis 14(8): 1224-3,2008; Patel et al., J Clin Virol 44(1):1-8, 2009). In immunocompromisedpatients, NoV is recognized as an important cause of chronicgastroenteritis, with long-term virus shedding and increased morbidityin this population (Ludwig, et al., J Med Virol 80(8): 1461-7, 2008;Henke-Gendo et al., J Clin Microbiol 47(9): 2855-62, 2009; Florescu etal., Pediatr Transplant 15(7): 718-21, 2011). In immunocompetentpatients the virus shedding after infection lasts for approximately 30days, while in immunocompromised patients virus shedding has beendetected for up to 3 years (Bok and Green, N Engl J Med 367(22):2126-32, 2012; Payne et al., N Engl J Med 368(12): 1121-30, 2013; Kirbyet al., J Med Virol, PubMed PMID: 24531909, 2014). It has been proposedthat long term virus shedding may contribute to the spread of the virus(Debbink et al., PLoS Pathog 8(10): e1002921, 2014; Debbink et al., JVirol., Mar. 192014).

The NoV genome is composed of a single-stranded positive-sense RNAmolecule that contains three open reading frames. The genome issurrounded by a non-enveloped capsid composed of the major capsidprotein, VP1, encoded by ORF2, and a minor structural protein, VP2,encoded by ORF3 (Green, Caliciviridae: The Noroviruses, Fields Virology,6th edition Knipe et al. (eds), published by Philadelphia: LippincottWilliams & Wilkins: 582-608, (ISBN-13: 978-1451105636), 2013).Crystallographic analysis showed that the NoV capsid is formed by 180molecules of VP1, organized into 90 dimers. Each VP1 monomer is dividedinto two domains designated shell (S) and protruding (P), linked by aflexible hinge. The P domain is further divided into P1 and P2subdomains, with P2 as the outermost domain exposed on the surface(Prasad et al., Science 286(5438): 287-90, 1999).

Noroviruses are divided into six major genogroups designated Genogroup(G)I through GVI. GI and GII contain the majority of NoV strainsassociated with human disease and are further divided into 9 and 21genotypes, respectively (Kroneman et al., Arch Virol 158(10): 2059-68,2013). The NoV GI.1 was the first genotype described, the GII.4 genotypehas been associated with the majority of global outbreaks since themid-1990s, when active surveillance with molecular diagnostic techniqueswas initiated (Zakikhany et al., PLoS One 7(7): e41625, 2012; Zheng etal., Virology 346(2): 312-23, 2006; Allen et al., Virol J 6: 150, 2009;Bok et al., J Virol 83(22): 11890-901, 2009; Patel et al., J Clin Virol44(1): 1-8, 2009; Lindesmith et al., J Virol 85(1): 231-42, 2011;Lindesmith et al., J Virol 87(5): 2803-13, 2013). A need remains forreagents that can be used to detect and treat NoV infections.

SUMMARY OF THE DISCLOSURE

Isolated monoclonal antibodies, such as V_(H)H monoclonal antibodies,are disclosed that specifically bind to a NoV polypeptide. NoVs includeGenogroup I and Genogroup II NoVs. NoVs include, for example, Norwalkvirus (NV) and MD2004 virus. The monoclonal antibodies can specificallybind Genogroup I or a Genogroup II NoV polypeptide. In specificnon-limiting examples, the monoclonal antibodies specifically bind VP1.The monoclonal antibodies can be neutralizing.

In some embodiments, the V_(H)H monoclonal antibody is a llamamonoclonal antibody. In some embodiments, the monoclonal antibody ishumanized. In some embodiments, the monoclonal antibody is chimeric.

In additional embodiments, disclosed are an isolated monoclonal antibodycomprising a heavy chain domain, wherein the heavy chain variable domaincomprises a heavy chain complementarity determining region (HCDR)1, anHCDR2 and an HCDR3, wherein the CDR3 comprises the amino acid sequenceset forth as one of: a) amino acids 96-109 of SEQ ID NO: 1; b) aminoacids 96-109 of SEQ ID NO: 2; c) amino acids 96-109 of SEQ ID NO: 3; d)amino acids 96-109 of SEQ ID NO: 4; e) amino acids 97-110 of SEQ ID NO:5; f) amino acids 97-111 of SEQ ID NO: 6; g) amino acids 97-111 of SEQID NO: 7; h) amino acids 97-111 of SEQ ID NO: 8; i) amino acids 96-112of SEQ ID NO: 9; j) amino acids 96-112 of SEQ ID NO: 10; k) amino acids96-114 of SEQ ID NO: 11; 1) amino acids 96-112 of SEQ ID NO: 12; m)amino acids 97-113 of SEQ ID NO: 13; n) amino acids 97-113 of SEQ ID NO:14; o) amino acids 97-114 of SEQ ID NO: 15; p) amino acids 96-107 of SEQID NO: 16; q) amino acids 97-113 of SEQ ID NO: 17; r) amino acids 97-114of SEQ ID NO: 18; s) amino acids 97-113 of SEQ ID NO: 19; t) amino acids96-111 of SEQ ID NO: 20; u) amino acids 96-102 of SEQ ID NO: 21; v)amino acids 96-110 of SEQ ID NO: 22; w) amino acids 95-104 of SEQ ID NO:23; x) amino acids 96-107 of SEQ ID NO: 24; y) amino acids 100-110 ofSEQ ID NO: 25; z) amino acids 96-117 of SEQ ID NO: 26; aa) amino acids97-108 of SEQ ID NO: 27; bb) amino acids 96-112 of SEQ ID NO: 28; cc)amino acids 97-115 of SEQ ID NO: 29; dd) amino acids 97-121 of SEQ IDNO: 30, and wherein the monoclonal antibody specifically binds aNorovirus polypeptide. The monoclonal antibody can be a V_(H)Hmonoclonal antibody.

Some embodiments provide nucleic acids encoding these antibodies,expression vectors including the nucleic acids, and isolated host cellsthat express the nucleic acids.

In further embodiments, methods are also disclosed for detecting thepresence of a NoV in a biological sample, such as Genogroup I andGenogroup II NoVs. These methods can detect a NoV infection in asubject. In a specific non-limiting example, NoV infection is a Norwalkvirus infection or an MD2004 virus infection. These methods includecontacting a biological sample of interest with an antibody disclosedherein, or an antigen binding fragment, and detecting binding of theantibody to the biological sample.

In other embodiments, methods are disclosed for treating and/orpreventing a NoV infection and/or disease. The methods can be used totreat and or prevent a Genogroup I and Genogroup II NoV infection, suchas a NV infection or a MD2004 virus infection in a subject. Thesemethods include administering to the subject a therapeutically effectiveamount of an antibody disclosed herein or antigen binding fragmentthereof, or a nucleic acid encoding the antibody or antigen bindingfragment, thereby treating or preventing the infection and/or disease.

The foregoing and other features and advantages of the invention willbecome more apparent from the following detailed description of severalembodiments which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1B. Llama immunization and immune response to NoV VLPs Norwalk(GI.1) and MD2004 (GII.4). The schedule for immunization (▾), samplecollection and final bleeding (♦) is shown. The evaluation of NoV Abresponse in serum during the time course of immunization is depicted.FIG. 1A. Antibody titers and number of ASC specific to VLPs of Norwalkstrain (GI.1) detected in the blood of the immunized llamas. FIG. 1B.Antibody titers and number of ASC specific to VLPs of MD2004 strain(GII.4) detected in the blood of the immunized llamas. Antibody titerswere measured by ELISA (lines) and the number of ASC were measured byELISPOT (bars) using recombinant NoV VLPs Norwalk and MD2004.

FIGS. 2A-2C. Blockade assays. Surrogate virus neutralization tests wereperformed using different sources of carbohydrates. FIG. 2A.Carbohydrate blockade assay. Synthetic carbohydrates H1 for Norwalk VLPs(GI.1) or H3 for MD145 VLPs (GII.4). FIG. 2B. PGM type III blockadeassay. PGM type III for Norwalk or MD2004 VLPs (GII.4). FIG. 2C. Saliva(antigen Ly+) blockade assay. Saliva for Norwalk or MD145 VLPs.Sigmoidal curves were fit to the mean percent control binding calculatedby comparing the amount of VLP bound to each source of carbohydrate inthe presence of V_(H)H pretreatment to the amount of VLP bound in theabsence of pretreatment.

FIGS. 3A-3B. Hemagglutination inhibition assay. The HAI titer of eachV_(H)H was defined as the lowest antibody concentration that completelyprevented NoV VLP-mediated hemagglutination of human RBC. Norwalk orNorovirus 2007 GI.1 VLPs hemagglutinated 0 Rh-RBC (FIG. 3A, GI.1 VLPs);MD2004 or MD145 GII.4 VLPs hemagglutinated B Rh+ (FIG. 3B, GII.4 VLPs).

FIGS. 4A-4B. Hyperimmune llama serum blocking assay. The percentage ofV_(H)H binding to hyperimmune serum pretreated VLPs was calculatedcompared to the positive control of each V_(H)H binding to the VLPswithout pretreatment. Bars represent the average of two independentassays. FIG. 4A. GI specific V_(H)Hs. FIG. 4B. GII specific V_(H)Hs.

FIGS. 5A-5B. Competition assay between V_(H)Hs. The images showimmunofluorescence staining of Vero cells expressing NoV VP1 fromNorwalk strain (FIG. 5A, GI specific V_(H)Hs 6.3 and 10.4) or MD2004strain (FIG. 5B, GII specific V_(H)Hs 1 and 5.4). Ten μg of an unlabeledV_(H)H together with 1 μl of Alexa Fluor 568 labeled V_(H)H wereincubated onto the fixed cells. A decrease in the fluorescent signalindicates the impaired binding of the labeled V_(H)H.

FIGS. 6A-6B. Epitope mapping of V_(H)H 7.5 FIG. 6A. ELISA of overlappingpeptides corresponding to the P domain of VP1. FIG. 6B. Linear epitopeof V_(H)H 7.5 within the VP1 dimer. Light grey color corresponds to P1subdomain, dark grey corresponds to P2 subdomain of VP1 and the putativeepitope is shown in lower left and right corner in very dark grey.

FIGS. 7A-7B. Epitope mapping of clone 16 specific for MD2004 strain.FIG. 7A. Immunofluorescence assay. Expression of MD2004 VP1 mutants inVero cells. The images show immunofluorescence staining of Vero cellstransfected with DNA constructs pCI carrying VP1 from wild type MD2004and 5 P domain mutants: E376Q; G340A, N368T, V3891I, V340/E376. V_(H)H16 binding was detected with rabbit anti-V_(H)H polyclonal serum andanti-rabbit IgG labeled with ALEXA FLUOR® 488. Labeled MAb TV20 directedto the S domain was used as positive control. FIG. 7B. Schematic diagramof the amino acids involved in V_(H)H 16 epitope: Light grey colorcorresponds to P1 subdomain, dark grey corresponds to P2 subdomain ofVP1 and the amino acids involved in the epitope are shown in dark grey.

FIG. 8. V_(H)Hs specific to GI or GII NoV VP1. Protein sequence of theV_(H)H antibody fragments selected by phage display. The four frameworkregions (FR) and the three complementary determining regions (CDR) areindicated with brackets; the two canonical cysteines are boxed. Thesequences shown are SEQ ID NOs: 1-30. The top bracket shows thesequences of antibodies that bind GII, and the bottom bracket shows thesequences of antibodies that bind GI.

FIG. 9A-9B. Western blots. FIG. 9A. Detection of NoV VLPs by WesternBlot by the V_(H)Hs. FIG. 9B. Western Blot detection of GII NoV VLPs bythe 7.5 V_(H)H.

SEQUENCE LISTING

The nucleic and amino acid sequences listed in the accompanying sequencelisting are shown using standard letter abbreviations for nucleotidebases, and three letter code for amino acids, as defined in 37 C.F.R.1.822. Only one strand of each nucleic acid sequence is shown, but thecomplementary strand is understood as included by any reference to thedisplayed strand. The Sequence Listing is submitted as an ASCII textfile in the form of the file named “4239-92920-13_Sequence_Listing.txt,”55.5 KB, which was created on Apr. 15, 2020, and is incorporated byreference herein.

DETAILED DESCRIPTION OF SEVERAL EMBODIMENTS

Camelid monoclonal antibodies, specifically a V_(H)H, or nanobody thatspecifically binds a NoV and/or a NoV protein are disclosed herein. Incertain embodiments, the monoclonal V_(H)H antibody or nanobody isproduced by the camelid, such as a llama, following immunization with aNoV, a NoV protein, or a peptide fragment thereof. Alternatively, thecamelid V_(H)H monoclonal antibody is engineered, i.e., produced byselection for example from a library of phage displaying appropriatelymutagenized V_(H)H monoclonal antibodies using panning procedures with aNoV and/or a protein component thereof as a target. Engineered V_(H)Hantibodies can further be customized by genetic engineering to have ahalf-life in a recipient subject, such as to increase half-life from 45minutes to two weeks. In a specific embodiment, the CDRs of a V_(H)Hmonoclonal antibody are grafted onto human framework sequences toproduced human antibody with the specificity of the V_(H)H.

A region of the camelid antibody which is the small single variabledomain identified as V_(H)H can be obtained by genetic engineering toyield a small protein having high affinity for a target, resulting in alow molecular weight antibody-derived protein known as a “camelidnanobody” or a “V_(H)H.” See U.S. Pat. No. 5,759,808, issued Jun. 2,1998; see also Stijlemans, et al., J Biol Chem 279: 1256-61, 2004;Dumoulin et al., Nature 424: 783-8, 2003; Pleschberger et al.,Bioconjugate Chem 14: 440-8, 2003; Cortez-Retamozo et al., Int J Cancer89: 456-62, 2002; and Lauwereys et al., EMBO J 17: 3512-3520, 1998.Without being bound by theory, a V_(H)H monoclonal antibody has amolecular weight approximately one-tenth that of a human IgG molecule,and the protein has a physical diameter of only a few nanometers. Oneconsequence of the small size is the ability of the V_(H)H monoclonalantibody to bind to antigenic sites that are functionally invisible tolarger antibody proteins, such that V_(H)H monoclonal antibodies areuseful as reagents to detect antigens that are otherwise cryptic usingclassical immunological techniques, and thus are of use as therapeuticagents. Thus yet another consequence of small size is that a camelidV_(H)H monoclonal antibody can inhibit as a result of binding to aspecific site in a groove or narrow cleft of a target protein, and hencecan serve in a capacity that more closely resembles the function of aclassical low molecular weight drug than that of a classical antibody.

Without being bound by theory, low molecular weight and compact sizefurther result in camelid V_(H)H monoclonal antibodies being extremelythermostable, stable to extreme pH and to proteolytic digestion, andpoorly antigenic. Another consequence is that camelid nanobodies readilymove from the circulatory system into tissues, and even cross theblood-brain barrier and can treat disorders that affect nervous tissue.Nanobodies can further facilitate drug transport across the blood brainbarrier (see U.S. Patent Application No. 20040161738). Further, thesemolecules can be fully expressed in prokaryotic cells such as E. coliand are expressed as fusion proteins with bacteriophage and arefunctional. Thus, the presently disclosed V_(H)H monoclonal antibodiesand antigen binding fragments are of use both as therapeutics and indiagnostic assays for a NoV.

Terms

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology maybe found in Lewin, Genes V, published by Oxford University Press, (ISBN0-19-854287-9), 1994; Kendrew et al. (eds.), The Encyclopedia ofMolecular Biology, published by Blackwell Science Ltd., (ISBN0-632-02182-9), 1994; and Meyers (ed.), Molecular Biology andBiotechnology: a Comprehensive Desk Reference, published by VCHPublishers, Inc., (ISBN 1-56081-569-8, 1995). Unless otherwiseexplained, all technical and scientific terms used herein have the samemeaning as commonly understood by one of ordinary skill in the art towhich this disclosure belongs.

Administration: The introduction of a composition into a subject by achosen route. Administration can be local or systemic. For example, ifthe chosen route is intravenous, the composition is administered byintroducing the composition into a vein of the subject. The chosen routecan, in some examples, be oral administration. In some examples adisclosed V_(H)H monoclonal antibody specific for a NoV, such as aNorwalk virus, is administered to a subject.

Agent: Any substance or any combination of substances that is useful forachieving an end or result; for example, a substance or combination ofsubstances useful for inhibiting a NoV infection in a subject. Agentsinclude, and are not limited to, proteins, nucleic acid molecules,compounds, small molecules, organic compounds, inorganic compounds, orother molecules of interest. An agent can include a therapeutic agent(such as an anti-viral agent), a diagnostic agent or a pharmaceuticalagent. In some embodiments, the agent is a polypeptide agent (such as aneutralizing antibody), or an anti-viral agent. The skilled artisan willunderstand that particular agents may be useful to achieve more than oneresult.

Amino acid substitution: The replacement of one amino acid in peptidewith a different amino acid.

Amplification: A technique that increases the number of copies of anucleic acid molecule (such as an RNA or DNA). An example ofamplification is the polymerase chain reaction, in which a biologicalsample is contacted with a pair of oligonucleotide primers, underconditions that allow for the hybridization of the primers to a nucleicacid template in the sample. The primers are extended under suitableconditions, dissociated from the template, and then re-annealed,extended, and dissociated to amplify the number of copies of the nucleicacid. The product of amplification can be characterized byelectrophoresis, restriction endonuclease cleavage patterns,oligonucleotide hybridization or ligation, and/or nucleic acidsequencing using standard techniques. Other examples of amplificationinclude strand displacement amplification, as disclosed in U.S. Pat. No.5,744,311; transcription-free isothermal amplification, as disclosed inU.S. Pat. No. 6,033,881; repair chain reaction amplification, asdisclosed in PCT Publication No. WO 90/01069; ligase chain reactionamplification, as disclosed in European Patent Publication EP-A-320 308;gap filling ligase chain reaction amplification, as disclosed in U.S.Pat. No. 5,427,930; and NASBA™ RNA transcription-free amplification, asdisclosed in U.S. Pat. No. 6,025,134.

Animal: Living multi-cellular vertebrate organisms, a category thatincludes, for example, mammals and birds. The term mammal includes bothhuman and non-human mammals, such as camelids. Similarly, the term“subject” includes both human and veterinary subjects.

Antibody: A polypeptide substantially encoded by an immunoglobulin geneor immunoglobulin genes, or antigen binding fragments thereof, whichspecifically binds and recognizes an analyte (antigen) such as, but notlimited to, a NoV polypeptide, such as a NV polypeptide. The antibodycan specifically bind VP1, or an immunogenic fragment thereof, forexample the P1 or P2 domain Immunoglobulin genes include the kappa,lambda, alpha, gamma, delta, epsilon and mu constant region genes, aswell as the myriad immunoglobulin variable region genes.

In several embodiments, in primates such as humans, a heavy and thelight chain variable domain of an antibody combine to specifically bindthe antigen. Generally, a naturally occurring primate (e.g., human) ormurine immunoglobulin has heavy (H) chains and light (L) chainsinterconnected by disulfide bonds. There are two types of light chain,lambda (λ) and kappa (κ). There are five main heavy chain classes (orisotypes) which determine the functional activity of an antibodymolecule: IgM, IgD, IgG, IgA and IgE. Primate antibodies can be classswitched.

In some embodiments, only the heavy chain variable domain is requiredfor antigen binding. For example, naturally occurring camelid antibodiesconsisting of a heavy chain only are functional and stable in theabsence of light chain (see, e.g., Hamers-Casterman et al., Nature,363:446-448, 1993; Sheriff et al., Nat. Struct. Biol., 3:733-736, 1996).Specifically, antibody proteins obtained from members of the camel anddromedary (Camelus bactrianus and Calelus dromaderius) family includingnew world members such as llama species (Lama paccos, Lama glama andLama vicugna) have been characterized with respect to size, structuralcomplexity and antigenicity for human subjects. Certain IgG antibodiesfrom this family of mammals as found in nature lack light chains, andare thus structurally distinct from the typical four chain quaternarystructure having two heavy and two light chains, for antibodies fromother animals. See PCT/EP93/02214 (WO 94/04678 published 3 Mar. 1994).

Light and heavy chain variable domains contain a “framework” regioninterrupted by three hypervariable regions, also called“complementarity-determining regions” or “CDRs” (see, e.g., Kabat etal., Sequences of Proteins of Immunological Interest, U.S. Department ofHealth and Human Services, 1991). The sequences of the framework regionsof different light or heavy chains are relatively conserved within aspecies. The framework region of an antibody, that is the combinedframework regions of the constituent light and heavy chains, serves toposition and align the CDRs in three-dimensional space. The CDRs areprimarily responsible for antigen binding.

References to “V_(H)” or “VH” refer to the variable region of animmunoglobulin heavy chain, including that of an antibody fragment.References to “V_(L)” or “VL” refer to the variable region of animmunoglobulin light chain, such as in a primate antibody. References to“V_(H)H” or “V_(H)H” refer to the variable region of a “heavy chainimmunoglobulin.”

The CDRs are typically referred to as CDR1, CDR2, and CDR3 (from theN-terminus to C-terminus), and are also typically identified by thechain in which the particular CDR is located. Thus, a V_(H) CDR3 islocated in the variable domain of the heavy chain of the antibody inwhich it is found, whereas a V_(L) CDR1 is the CDR1 from the variabledomain of the light chain of the antibody in which it is found. Lightchain CDRs are sometimes referred to as CDR L1, CDR L2, and CDR L3.Heavy chain CDRs are sometimes referred to as CDR H1, CDR H2, and CDRH3. V_(H)H monoclonal antibodies have only a heavy chain, and thusinclude only one CDR1, CDR2 and CDR3. Generally, the CDR3 is primarilyresponsible for antigen specificity. The sequences of the frameworkregions of different light or heavy chains are relatively conservedwithin a species. The framework region of an antibody, that is thecombined framework regions of the constituent light and heavy chains,serves to position and align the CDRs in three-dimensional space.

Camelids produce a unique antibody molecule. Some IgG subtypes of thellamas lack the light chains and the CH1 domain and are called heavychain antibodies.

Each light and heavy chain of any antibody, contains constant domainsand variable domains. Light and heavy chain variable domains, are namedV_(L) and V_(H) respectively, while the variable domain of a heavy chainantibody is called V_(H)H. The V_(H)H is composed of only onepolypeptide chain of 15 kDa and is considered the smallest known naturaldomain with full antigen-binding capacity.

Any variable domain includes in an N- to C-direction, the followingstructural regions: N-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-C, wherein FRdenotes a framework region amino acid sequence and CDR denotes acomplementary determining region amino acid sequence (see, e.g., Kabatet al., Sequences of Proteins of Immunological Interest, U.S. Departmentof Health and Human Services, 1991). In specific non-limiting examples,the CDR3 comprises a llama CDR3 V_(H)H domain amino acid sequence; andwherein the antibody binds to Norovirus.

The extent of the framework region and CDRs have been defined (see,Kabat et al., Sequences of Proteins of Immunological Interest, U.S.Department of Health and Human Services, 1991, which is herebyincorporated by reference in its entirety). The CDRs of the heavy chainvariable domain are located at residues 31-35 (CDR-H1), residues 50-65(CDR-H2) and residues 95-102 (CDR-H3) according to the Kabat numberingsystem. However, according to Chothia (Chothia and Lesk, J. Mol. Biol.,196: 901-17, 1987), the loop equivalent to CDR-H1 extends from residue26 to residue 32. Thus “CDR-H1”, as used herein, comprises residues 26to 33, as described by a combination of the Kabat numbering system andChothia's topological loop definition. In antibodies (such as primateantibodies) that include a light chain, such as a primate antibody, theCDRs of the light chain variable domain are located at residues 24-34(CDR-L1), residues 50-56 (CDR-L2) and residues 89-97 (CDR-L3) accordingto the Kabat numbering system. Lefranc, et al. (“IMGT unique numberingfor immunoglobulin and T cell receptor variable domains and Igsuperfamily V-like domains,” Dev. Comp. Immunol., 27:55-77, 2003)discloses the “IMGT” numbering scheme for CDRs. The Kabat database isnow maintained online. The location of camelid CDRs can also bedetermined (see, for example, Sircar et al., J. Immunol. 186: 6357-6367,2011); a program to determine camelid antibody structure, theRosettaAntibody program, is available on the internet.

A “monoclonal antibody” is an antibody produced by a single clone ofB-lymphocytes or by a cell into which the heavy chain gene (andoptionally a light chain gene, such as for a primate antibody) of asingle antibody have been transfected.

In some embodiments, the V_(H)H molecules can be produced as recombinantmonoclonal antibodies or antigen binding fragments in differentexpression platforms, avoiding the use of hybridomas and mice. V_(H)Hmonoclonal antibodies can be humanized monoclonal antibodies. In someembodiments, monoclonal antibodies can be chimeric antibodies.

A “chimeric” antibody is an antibody which includes sequences from twodifferent antibodies, which typically are of different species. In someexamples, a chimeric antibody includes one or more CDRs and/or frameworkregions from one llama V_(H)H and CDRs and/or framework regions fromanother llama V_(H)H. In some embodiments, a chimeric antibody comprisesheavy and/or light chain variable regions derived from a first speciesand heavy and/or light chain constant regions derived from a secondspecies. In some embodiments, the variable and constant regions of thelight chain are derived from a first species (for example, a primate,such as a human) while the variable region of the heavy chain is derivedfrom a second species (for example, a llama) and the constant region ofthe heavy chain is derived from the first species.

A “humanized” antibody is an antibody including a human framework regionand one or more CDRs from a non-human (such as a camelid, chimpanzee,mouse, rat, llama or synthetic) immunoglobulin. The non-human antibodyproviding the CDRs is termed a “donor,” and the human antibody providingthe framework is termed an “acceptor.” In one embodiment, all the CDRsare from the donor antibody in a humanized antibody. Constant regionsneed not be present, but if they are, they must be substantiallyidentical to human antibody constant regions, such as at least about85-90%, such as about 95% or more identical. Hence, all parts of ahumanized antibody, except possibly the CDRs, are substantiallyidentical to corresponding parts of natural human antibody sequences. A“humanized antibody” can include a humanized light chain and a humanizedheavy chain. A humanized antibody binds to the same antigen as the donorantibody that provides the CDRs. The acceptor framework of a humanizedantibody may have a limited number of substitutions by amino acids takenfrom the donor framework. Humanized or other monoclonal antibodies canhave additional conservative amino acid substitutions which havesubstantially no effect on antigen binding or other immunoglobulinfunctions. Humanized immunoglobulins can be constructed by means ofgenetic engineering (for example, see U.S. Pat. No. 5,585,089).

AV_(H)H antibody is easily humanized, although camelid antibodies havehigh homology with the human V_(H) domain. In some embodiments, ahumanized V_(H)H has amino acid mutations in specific sites of the FRsregions, for example, see PCT Publication No, WO2013030604 A1,incorporated by reference herein).

A “neutralizing antibody” is an antibody which reduces the infectioustiter of an infectious agent by binding to a specific antigen on theinfectious agent. In some examples the infectious agent is a virus. Insome examples, an antibody that is specific for a NoV, such as a Norwalkvirus, neutralizes the infectious titer of the virus or demonstratesability to block norovirus VLPs binding to ligands in a surrogateneutralization assays.

Two chain antibodies, such as primate antibodies exist, for example, asintact immunoglobulins and as a number of well-characterized fragmentsproduced by digestion with various peptidases. For instance, with regardto two-chain antibodies, such as primate (e.g., human) and murineantibodies, Fabs, Fvs, scFvs that specifically bind to a NoVpolypeptide, for example VP1, or fragments of this polypeptide, arespecific binding agents. A scFv protein is a fusion protein in which alight chain variable region of an immunoglobulin and a heavy chainvariable region of an immunoglobulin are bound by a linker, while indsFvs, the chains have been mutated to introduce a disulfide bond tostabilize the association of the chains. The term also includesgenetically engineered forms such as chimeric antibodies andheteroconjugate antibodies such as bispecific antibodies. See also,Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford,Ill.); Kuby, Immunology, 3^(rd) Ed., W.H. Freeman & Co., New York, 1997.

Conventional antibody fragments of two chain molecules include, but arenot limited to, the following: (1) Fab, the fragment which contains amonovalent antigen-binding fragment of an antibody molecule produced bydigestion of whole antibody with the enzyme papain to yield an intactlight chain and a portion of one heavy chain; (2) Fab′, the fragment ofan antibody molecule obtained by treating whole antibody with pepsin,followed by reduction, to yield an intact light chain and a portion ofthe heavy chain; two Fab′ fragments are obtained per antibody molecule;(3) (Fab′)₂, the fragment of the antibody obtained by treating wholeantibody with the enzyme pepsin without subsequent reduction; (4)F(ab′)₂, a dimer of two Fab′ fragments held together by two disulfidebonds; (5) Fv, a genetically engineered fragment containing the variableregion of the light chain and the variable region of the heavy chainexpressed as two chains; and (6) single chain antibody (“SCA”), agenetically engineered molecule containing the variable region of thelight chain, the variable region of the heavy chain, linked by asuitable polypeptide linker as a genetically fused single chainmolecule.

Camelids produce a unique antibody molecule. Some IgG subtypes of thellamas lack the light chain and are called heavy chain antibodies; thevariable domain of these antibodies is called V_(H)H and is comprised ofonly one polypeptide chain. The V_(H)H domain is a molecule of 15 kDathat is considered the smallest known natural domain with fullantigen-binding capacity. The DNA encoding the V_(H)H region can beobtained and modified by genetic engineering to yield a small proteinhaving high affinity for a target, resulting in a low molecular weightantibody-derived protein known as a camelid “nanoantibody” or“nanobody”. There is no Fc region nor V_(L) domain in a recombinantV_(H)H. See U.S. Pat. No. 5,759,808 issued Jun. 2, 1998; see alsoStijlemans et al., J Biol Chem 279: 1256-1261, 2004; Dumoulin et al.,Nature 424: 783-788, 2003; Pleschberger et al., Bioconjugate Chem 14:440-448, 2003; Cortez-Retamozo et al. Int J Cancer 89: 456-62, 2002; andLauwereys et al., EMBO J 17: 3512-3520, 1998.

Antigen binding fragments of an antibody can be produced by themodification of whole antibodies or those synthesized de novo usingrecombinant DNA methodologies. In some examples, the term antibodyincludes the amino acid sequences of one or more of the CDRs from theantibody grafted onto a scaffold.

Antigen: A compound, composition, or substance that can stimulate theproduction of antibodies or a T cell response in an animal, includingcompositions that are injected or absorbed into an animal. An antigenreacts with the products of specific humoral or cellular immunity,including those induced by heterologous antigens, such as the disclosedantigens. “Epitope” or “antigenic determinant” refers to the region ofan antigen to which B and/or T cells respond. In one embodiment, T cellsrespond to the epitope, when the epitope is presented in conjunctionwith an MHC molecule. Epitopes can be formed both from contiguous aminoacids or noncontiguous amino acids juxtaposed by tertiary folding of aprotein. Epitopes formed from contiguous amino acids are typicallyretained on exposure to denaturing solvents whereas epitopes formed bytertiary folding are typically lost on treatment with denaturingsolvents. An epitope typically includes at least 3, and more usually, atleast 5, about 9, or about 8-10 amino acids in a unique spatialconformation. Methods of determining spatial conformation of epitopesinclude, for example, x-ray crystallography and nuclear magneticresonance.

Examples of antigens include, but are not limited to, peptides, lipids,polysaccharides, and nucleic acids containing antigenic determinants,such as those recognized by an immune cell. In some examples, antigensinclude peptides derived from a pathogen of interest. Exemplarypathogens include bacteria, fungi, viruses and parasites. In someembodiments, an antigen is derived from a NoV, such as a Norwalk virusor a MD2004 virus. In some embodiments, the antigen is a NoV VP1polypeptide or antigenic fragment thereof, such as a P1 or P2 domain.

A “target epitope” is a specific epitope on an antigen that specificallybinds an antibody of interest, such as a monoclonal antibody. In someexamples, a target epitope includes the amino acid residues that contactthe antibody of interest, such that the target epitope can be selectedby the amino acid residues determined to be in contact with theantibody.

Binding affinity: Affinity of an antibody, such as a V_(H)H monoclonalantibody, or antigen binding fragment thereof for an antigen. In oneembodiment, affinity is calculated by a modification of the Scatchardmethod described by Frankel et al., Mol. Immunol., 16:101-106, 1979. Inanother embodiment, binding affinity is measured by an antigen/antibodydissociation rate. In yet another embodiment, a high binding affinity ismeasured by a competition radioimmunoassay or by Plasmon resonance in aBIOCORE. In several examples, a high binding affinity is at least about1×10⁻⁸ M. In other embodiments, a high binding affinity is at leastabout 1.5×10⁻⁸, at least about 2.0×10⁻⁸, at least about 2.5×10⁻⁸, atleast about 3.0×10⁻⁸, at least about 3.5×10⁻⁸, at least about 4.0×10⁻⁸,at least about 4.5×10⁻⁸, or at least about 5.0×10⁻⁸ M.

The antigen specificity and affinity of V_(H)H antibodies from immunelibraries are of good quality. Kinetick (k)on and koff rate constantsare generally in the range of 10⁵ to 10⁶ M⁻¹ s⁻¹ and 10⁻² to 10⁻¹ s⁻¹,respectively, such that low nanomolar or even picomolar equilibriumdissociation constants are obtained. Such affinity parameters areexcellent for most applications (Muyldermans, Annu. Rev. Biochem. 2013.82:775-97, 2013).

Capsid protein (VP1): A capsid polypeptide that is encoded by openreading frame (ORF) 2 of the NoV genome; the polypeptide itselfassembles to form an icosahedral capsid. When the protein is 530 aminoacids in length, the shell (S) domain (amino acids 1-225) containselements necessary for the formation of the icosahedron. The Protrudingdomain (P, amino acids 225-530) is divided into sub-domains P1 (aminoacids 226-278 (P1 subdomain 1) and 406-530 (P1 subdomain 2) and P2(amino acids 279-405). The P domain interacts in dimeric contacts thatincrease the stability of the capsid and form the protrusions on thevirion. The P2 domain is hypervariable. An exemplary VP1 is provided inUNIPROT Accession No. Q83884 (for example, CAPSD_NVN68, Oct. 3, 2012),which is incorporated herein by reference.

Clonal variant: Any sequence, which differs by one or more nucleotidesor amino acids, in presence of V region with identical mutationscompared to the germline, identical VDJ or VJ gene usage, and identicalD and J length. The “germline” sequence is intended to be the sequencecoding for the antibody/immunoglobulin (or of any fragment thereof)deprived of mutations, for example somatic mutations. The percentage ofhomology represents an indication of the mutational events which anytype of heavy chain portion undergoes after contact with an antigen.

Computer readable media: Any medium or media, which can be read andaccessed directly by a computer, so that the media is suitable for usein a computer system. Such media include, but are not limited to:magnetic storage media such as floppy discs, hard disc storage mediumand magnetic tape; optical storage media such as optical discs orCD-ROM; electrical storage media such as RAM and ROM; and hybrids ofthese categories such as magnetic/optical storage media.

Conjugate: A complex of two molecules linked together, for example,linked together by a covalent bond. In one embodiment, an antibody islinked to an effector molecule; for example, an antibody, such as aV_(H)H monoclonal antibody, that specifically binds to a NoVpolypeptide, such as a NV polypeptide, covalently linked to an effectormolecule or to a toxin. The linkage can be by chemical or recombinantmeans. In one embodiment, the linkage is chemical, wherein a reactionbetween the antibody moiety and the effector molecule has produced acovalent bond formed between the two molecules to form one molecule. Apeptide linker (short peptide sequence) can optionally be includedbetween the antibody and the effector molecule. Because conjugates canbe prepared from two molecules with separate functionalities, such as anantibody and an effector molecule, they are also sometimes referred toas “chimeric molecules.” In one embodiment, an antibody linked to aneffector molecule is further joined to a lipid or other molecule to aprotein or peptide to increase its half-life in the body.

Contacting: Placement in direct physical association; includes both insolid and liquid form, which can take place either in vivo or in vitro.Contacting includes contact between one molecule and another molecule,for example the amino acid on the surface of one polypeptide, such as anantigen, that contacts another polypeptide, such as an antibody.Contacting can also include contacting a cell for example by placing anantibody in direct physical association with a cell.

Control: A reference standard. In some embodiments, the control is asample obtained from a healthy patient. In other embodiments, thecontrol is a tissue sample obtained from a patient diagnosed with a NoVinfection, such as a Norwalk virus infection that serves as a positivecontrol. In still other embodiments, the control is a historical controlor standard reference value or range of values (such as a previouslytested control sample, such as a group of infected patients with knownprognosis or outcome, or group of samples that represent baseline ornormal values).

A difference between a test sample and a control can be an increase orconversely a decrease. The difference can be a qualitative difference ora quantitative difference, for example a statistically significantdifference. In some examples, a difference is an increase or decrease,relative to a control, of at least about 5%, such as at least about 10%,at least about 20%, at least about 30%, at least about 40%, at leastabout 50%, at least about 60%, at least about 70%, at least about 80%,at least about 90%, at least about 100%, at least about 150%, at leastabout 200%, at least about 250%, at least about 300%, at least about350%, at least about 400%, at least about 500%, or greater than 500%.

Detectable marker: A detectable molecule (also known as a label) that isconjugated directly or indirectly to a second molecule, such as anantibody, to facilitate detection of the second molecule. For example,the detectable marker can be capable of detection by ELISA,spectrophotometry, flow cytometry, microscopy or diagnostic imagingtechniques (such as CT scans, MRIs, ultrasound, fiberoptic examination,and laparoscopic examination). Non-limiting examples of detectablemarkers include fluorophores, fluorescent proteins, chemiluminescentagents, enzymatic linkages, radioactive isotopes and heavy metals orcompounds (for example super paramagnetic iron oxide nanocrystals fordetection by MRI). In one example, a “labeled antibody” refers toincorporation of another molecule in the antibody. For example, thelabel is a detectable marker, such as the incorporation of aradiolabeled amino acid or attachment to a polypeptide of biotinylmoieties that can be detected by marked avidin (for example,streptavidin containing a fluorescent marker or enzymatic activity thatcan be detected by optical or colorimetric methods). Various methods oflabeling polypeptides and glycoproteins are known in the art and may beused. Examples of labels for polypeptides include, but are not limitedto, the following: radioisotopes or radionuclides (such as ³⁵S or ¹³¹I),fluorescent labels (such as fluorescein isothiocyanate (FITC),rhodamine, lanthanide phosphors ALEXA FLUOR®), enzymatic labels (such ashorseradish peroxidase, beta-galactosidase, luciferase, alkalinephosphatase), chemiluminescent markers, biotinyl groups, predeterminedpolypeptide epitopes recognized by a secondary reporter (such as aleucine zipper pair sequences, binding sites for secondary antibodies,metal binding domains, epitope tags), or magnetic agents, such asgadolinium chelates. In some embodiments, labels are attached by spacerarms of various lengths to reduce potential steric hindrance. Methodsfor using detectable markers and guidance in the choice of detectablemarkers appropriate for various purposes are discussed for example inSambrook et al. (Molecular Cloning: A Laboratory Manual, Cold SpringHarbor, N.Y., 1989) and Ausubel et al. (In Current Protocols inMolecular Biology, John Wiley & Sons, New York, 1998).

Detecting: To identify the existence, presence, or fact of something.General methods of detecting are known to the skilled artisan (see, forexample, U.S. Pat. No. 7,635,476) and may be supplemented with theprotocols and reagents disclosed herein. For example, included hereinare methods of detecting a cell that expresses a NoV polypeptide, suchas a Norwalk virus polypeptide, for example, VP1, in a subject. In someembodiments, the peptide can be VP1.

Effector molecule: The portion of a chimeric molecule that is intendedto have a desired effect on a cell to which the chimeric molecule istargeted. Effector molecule is also known as an effector moiety,therapeutic agent, or diagnostic agent, or similar terms.

Epitope: An antigenic determinant. These are particular chemical groupsor peptide sequences on a molecule that are antigenic, i.e. that elicita specific immune response. An antibody specifically binds a particularantigenic epitope on a polypeptide. In some examples a disclosedantibody specifically binds to an epitope on the surface of VP1 from aNoV, such as the P2 subdomain.

Framework Region: Amino acid sequences interposed between CDRs. The termincludes variable light and variable heavy framework regions. Theframework regions serve to hold the CDRs in an appropriate orientationfor antigen binding.

Fc polypeptide: The polypeptide comprising the constant region of anantibody excluding the first constant region immunoglobulin domain. Fcregion generally refers to the last two constant region immunoglobulindomains of IgA, IgD, and IgG, and the last three constant regionimmunoglobulin domains of IgE and IgM. An Fc region may also includepart or all of the flexible hinge N-terminal to these domains. For IgAand IgM, an Fc region may or may not comprise the tailpiece, and may ormay not be bound by the J chain. For IgG, the Fc region comprisesimmunoglobulin domains Cgamma2 and Cgamma3 (Cγ2 and Cγ3) and the lowerpart of the hinge between Cgamma1 (Cγ1) and Cγ2. Although the boundariesof the Fc region may vary, the human IgG heavy chain Fc region isusually defined to comprise residues C226 or P230 to itscarboxyl-terminus, wherein the numbering is according to the EU index asin Kabat. For IgA, the Fc region comprises immunoglobulin domainsCalpha2 and Calpha3 (Cα2 and Cα3) and the lower part of the hingebetween Calpha1 (Cα1) and Cα2. Encompassed within the definition of theFc region are functionally equivalent analogs and variants of the Fcregion. A functionally equivalent analog of the Fc region may be avariant Fc region, comprising one or more amino acid modificationsrelative to the wild-type or naturally existing Fc region. Variant Fcregions will possess at least 50% homology with a naturally existing Fcregion, such as about 80%, and about 90%, or at least about 95%homology. Functionally equivalent analogs of the Fc region may compriseone or more amino acid residues added to or deleted from the N- orC-termini of the protein, such as no more than 30 or no more than 10additions and/or deletions. Functionally equivalent analogs of the Fcregion include Fc regions operably linked to a fusion partner.Functionally equivalent analogs of the Fc region must comprise themajority of all of the Ig domains that compose Fc region as definedabove; for example IgG and IgA Fc regions as defined herein mustcomprise the majority of the sequence encoding CH₂ and the majority ofthe sequence encoding CH₃. Thus, the CH₂ domain on its own, or the CH₃domain on its own, are not considered Fc region. The Fc region may referto this region in isolation, or this region in the context of an Fcfusion polypeptide.

Heavy chain antibody: Antibodies obtained from members of the camel anddromedary (Camelus bactrianus and Calelus dromaderius) family includingnew world members such as llama species (Lama pacos, Lama glama and Lamavicugna), specifically certain IgG antibodies (of the IgG₂ and IgG₃isotypes) that lack light chains and are thus structurally distinct fromthe typical four chain quaternary structure having two heavy and twolight chains (See PCT Publication No. WO 94/04678, incorporated hereinby reference). Naturally occurring camelid antibodies composed only byheavy chains are functional and stable in the absence of light chains(see, e.g., Hamers-Casterman et al., Nature, 363:446-448, 1993; Sheriffet al., Nat. Struct. Biol., 3:733-736, 1996). The heavy chain antibodiesare composed of two constant domains in the Fc and one variable domaincalled V_(H)H. These antibodies lack the CH1 domain of conventionalantibodies (Muyldermans, Annu. Rev. Biochem. 2013. 82:775-97, 2013).

Hemagglutination: Human erythrocytes are a natural source of humanhisto-blood group antigens (HBGA) ligands. NoV recombinant VLPs havehemagglutination activity through binding of HBGAs present in thesurface of human red blood cells. Antibodies directed to the HBGAbinding sites of the virus can inhibit hemagglutination(hemagglutination inhibition activity, HAI). An HAI assay can be used asan alternative to the HBGA blocking assay in order to quantitateneutralizing antibodies in a sample (Czako et al. Clinical and VaccineImmunology, 19(2), 284-7, 2012).

Host cells: Cells in which a vector can be propagated and its DNAexpressed, for example a disclosed antibody can be expressed in a hostcell. The cell may be prokaryotic or eukaryotic. The term also includesany progeny of the subject host cell. It is understood that all progenymay not be identical to the parental cell since there may be mutationsthat occur during replication. However, such progeny are included whenthe term “host cell” is used.

IgG: A polypeptide belonging to the class or isotype of antibodies thatare substantially encoded by a recognized immunoglobulin gamma gene. Inhumans, this class comprises IgG₁, IgG₂, IgG₃, and IgG₄. In camelids,this class comprises IgG₁, IgG₂, and IgG₃.

Immune complex: The binding of antibody to a soluble antigen forms animmune complex. The formation of an immune complex can be detectedthrough conventional methods known to the skilled artisan, for instanceimmunohistochemistry, immunoprecipitation, flow cytometry,immunofluorescence microscopy, ELISA, immunoblotting (for example,Western blot), magnetic resonance imaging, CT scans, X-ray and affinitychromatography Immunological binding properties of selected antibodiesmay be quantified using methods well known in the art.

Immunoadhesin: A molecular fusion of a protein with the Fc region of animmunoglobulin, wherein the immunoglobulin retains specific properties,such as Fc receptor binding and increased half-life. An Fc fusioncombines the Fc region of an immunoglobulin with a fusion partner, whichin general can be any protein, polypeptide, peptide, or small molecule.In one example, an immunoadhesin includes the hinge, CH₂, and CH₃domains of the immunoglobulin gamma 1 heavy chain constant region. Inanother example, the immunoadhesin includes the CH₂, and CH₃ domains ofan IgG.

Immunologically reactive conditions: Includes reference to conditionswhich allow an antibody raised against a particular epitope to bind tothat epitope to a detectably greater degree than, and/or to thesubstantial exclusion of, binding to substantially all other epitopes.Immunologically reactive conditions are dependent upon the format of theantibody binding reaction and typically are those utilized inimmunoassay protocols or those conditions encountered in vivo. SeeHarlow & Lane, supra, for a description of immunoassay formats andconditions. The immunologically reactive conditions employed in themethods are “physiological conditions” which include reference toconditions (e.g., temperature, osmolarity, pH) that are typical inside aliving mammal or a mammalian cell. While it is recognized that someorgans are subject to extreme conditions, the intra-organismal andintracellular environment normally lies around pH 7 (e.g., from pH 6.0to pH 8.0, more typically pH 6.5 to 7.5), contains water as thepredominant solvent, and exists at a temperature above 0° C. and below50° C. Osmolarity is within the range that is supportive of cellviability and proliferation.

Inhibiting or treating a disease: Inhibiting the full development of adisease or condition, for example, in a subject who is at risk for adisease such a NoV infection. “Treatment” refers to a therapeuticintervention that ameliorates a sign or symptom of a disease orpathological condition after it has begun to develop. The term“ameliorating,” with reference to a disease or pathological condition,refers to any observable beneficial effect of the treatment. Thebeneficial effect can be evidenced, for example, by a delayed onset ofclinical symptoms of the disease in a susceptible subject, a reductionin severity of some or all clinical symptoms of the disease, a slowerprogression of the disease, a reduction in the viral load, animprovement in the overall health or well-being of the subject, or byother parameters well known in the art that are specific to theparticular disease. A “prophylactic” treatment is a treatmentadministered to a subject who does not exhibit signs of a disease orexhibits only early signs for the purpose of decreasing the risk ofdeveloping pathology.

Isolated: An “isolated” biological component (such as a cell, forexample a B-cell, a nucleic acid, peptide, protein, heavy chain domainor antibody) has been substantially separated, produced apart from, orpurified away from other biological components in the cell of theorganism in which the component naturally occurs, such as, otherchromosomal and extrachromosomal DNA and RNA, and proteins. Nucleicacids, peptides and proteins which have been “isolated” thus includenucleic acids and proteins purified by standard purification methods.The term also embraces nucleic acids, peptides, and proteins prepared byrecombinant expression in a host cell as well as chemically synthesizednucleic acids. In some examples an antibody, such as an antibodyspecific for a NoV polypeptide can be isolated, for example isolatedfrom a subject infected with the virus.

K_(d): The dissociation constant for a given interaction, such as apolypeptide ligand interaction or an antibody antigen interaction. Forexample, for the bimolecular interaction of an antibody (such as any ofthe antibodies disclosed herein) and an antigen (such as a NoVpolypeptide, for example a Norwalk virus polypeptide) it is theconcentration of the individual components of the bimolecularinteraction divided by the concentration of the complex.

Label: A detectable compound or composition that is conjugated directlyor indirectly to another molecule, such as an antibody or a protein, tofacilitate detection of that molecule. Specific, non-limiting examplesof labels include fluorescent tags, enzymatic linkages, and radioactiveisotopes. In some examples, a disclosed antibody is labeled.

Neutralizing antibody: An antibody which reduces the infectious titer ofan infectious agent by binding to a specific antigen on the infectiousagent. In some examples the infectious agent is a virus, such as a NoV,for example a Genogroup I or Genogroup II NoV, such as a Norwalk virusor MD2004 virus. In some examples, an antibody that is specific for aNoV polypeptide neutralizes the infectious titer of the virus. In someexamples, an antibody specific for NoV VP1 neutralizes the infectioustiter of the virus. In vitro assays for neutralization are known in theart. Thus, in some non-limiting examples, an assay for neutralizationactivity is blocking the binding of NoV-like particles (VLPs) to HBGAsynthetic carbohydrates, for example H1 or H3 type HBGA, in a dosedependent manner. In other non-limiting examples an assay forneutralization activity is blocking the binding of NoV-VLPs to piggastric mucin or saliva, in a dose dependent manner. In othernon-limiting examples, an assay for neutralization is the inhibition ofhemagglutination activity.

With regard to an antigen from a pathogen, such as a virus, a “broadlyneutralizing” antibody can bind to and inhibit the function of anantigen from more than one class and/or subclass of the pathogen. Forexample, with regard to a NoV, the antibody can bind to and inhibit thefunction of an antigen, such as a viral protein, from more than onegenotype of NoV, including, but not limited to, NV (GI.1), P7-587(GI.1),Desert Shield virus (GI.3), Hawaii virus (GII.1), Snow Mountain virus(GII.2), Henryton virus (GII.2), CHDC2005 virus (GII.3), Toronto 24virus (GII.3), CHDC5261 virus (GII.3), CHDC4031 virus (GII.3),Maizuru2000 virus (GII.3), Aus2001, Aus2007 and Aus2008 virus (GII.3),CHDC32 virus (GII.3), CHDC4871 virus (GII.4), Rockville virus (GII.4),MD145 virus (GII.4), MD2004 virus (GII.4), HS191 virus (GII.4), Bethesdavirus (GII.6), DC119 virus (GII.7), M7 virus (GII.14) or viruses frommore than one genogroup (GI-GV). In one embodiment, broadly neutralizingantibodies to NoVs are distinct from other antibodies in that theyneutralize a high percentage of the many types of NoVs.

Nanoantibody or nanobody: The variable domain of a heavy chain antibody,called V_(H)H. A nanobody is comprised of only one polypeptide chain andis considered the smallest known natural domain with fullantigen-binding capacity (15 kDa). The DNA encoding the V_(H)H regionmay be obtained and modified by genetic engineering to yield a smallrecombinant protein having high affinity for a target, resulting in alow molecular weight antibody-derived protein known as a camelid“nanoantibody” or “nanobody” due to the small size. See U.S. Pat. No.5,759,808 issued Jun. 2, 1998; see also Stijlemans et al., J Biol Chem279: 1256-1261, 2004; Dumoulin et al., Nature 424: 783-788, 2003;Pleschberger et al., Bioconjugate Chem 14: 440-448, 2003;Cortez-Retamozo et al. Int J Cancer 89: 456-62, 2002; and Lauwereys etal., EMBO J 17: 3512-3520, 1998.

As noted above, a V_(H)H includes in an N- to C-direction, the followingstructural domains regions: N-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-C, whereinFR denotes a framework region amino acid sequence and CDR denotes acomplementary determining region amino acid sequence (see, e.g., Kabatet al., Sequences of Proteins of Immunological Interest, U.S. Departmentof Health and Human Services, 1991). In specific non-limiting examples,the CDR3 comprises a llama CDR3 V_(H)H domain amino acid sequence; andwherein the antibody binds to NoV. V_(H)H monoclonal antibodies haveonly a heavy chain (they do not include a light chain), and thus includeonly one CDR1, CDR2 and CDR3. Generally, the CDR3 is primarilyresponsible for antigen specificity.

Nucleic acid: A polymer composed of nucleotide units (ribonucleotides,deoxyribonucleotides, related naturally occurring structural variants,and synthetic non-naturally occurring analogs thereof) linked viaphosphodiester bonds, related naturally occurring structural variants,and synthetic non-naturally occurring analogs thereof. Thus, the termincludes nucleotide polymers in which the nucleotides and the linkagesbetween them include non-naturally occurring synthetic analogs, such as,for example and without limitation, phosphorothioates, phosphoramidates,methyl phosphonates, chiral-methyl phosphonates, 2-O-methylribonucleotides, peptide-nucleic acids (PNAs), and the like. Suchpolynucleotides can be synthesized, for example, using an automated DNAsynthesizer. The term “oligonucleotide” typically refers to shortpolynucleotides, generally no greater than about 50 nucleotides. It willbe understood that when a nucleotide sequence is represented by a DNAsequence (i.e., A, T, G, C), this also includes an RNA sequence (i.e.,A, U, G, C) in which “U” replaces “T.”

Conventional notation is used herein to describe nucleotide sequences:the left-hand end of a single-stranded nucleotide sequence is the5′-end; the left-hand direction of a double-stranded nucleotide sequenceis referred to as the 5′-direction. The direction of 5′ to 3′ additionof nucleotides to nascent RNA transcripts is referred to as thetranscription direction. The DNA strand having the same sequence as anmRNA is referred to as the “coding strand;” sequences on the DNA strandhaving the same sequence as an mRNA transcribed from that DNA and whichare located 5′ to the 5′-end of the RNA transcript are referred to as“upstream sequences;” sequences on the DNA strand having the samesequence as the RNA and which are 3′ to the 3′ end of the coding RNAtranscript are referred to as “downstream sequences.”

“cDNA” refers to a DNA that is complementary or identical to an mRNA, ineither single stranded or double stranded form.

“Encoding” refers to the inherent property of specific sequences ofnucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, toserve as templates for synthesis of other polymers and macromolecules inbiological processes having either a defined sequence of nucleotides(i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and thebiological properties resulting therefrom. Thus, a gene encodes aprotein if transcription and translation of mRNA produced by that geneproduces the protein in a cell or other biological system. Both thecoding strand, the nucleotide sequence of which is identical to the mRNAsequence and is usually provided in sequence listings, and non-codingstrand, used as the template for transcription, of a gene or cDNA can bereferred to as encoding the protein or other product of that gene orcDNA. Unless otherwise specified, a “nucleotide sequence encoding anamino acid sequence” includes all nucleotide sequences that aredegenerate versions of each other and that encode the same amino acidsequence. Nucleotide sequences that encode proteins and RNA may includeintrons.

“Recombinant nucleic acid” refers to a nucleic acid having nucleotidesequences that are not naturally joined together. This includes nucleicacid vectors comprising an amplified or assembled nucleic acid which canbe used to transform a suitable host cell. A host cell that comprisesthe recombinant nucleic acid is referred to as a “recombinant hostcell.” The gene is then expressed in the recombinant host cell toproduce, e.g., a “recombinant polypeptide.” A recombinant nucleic acidmay serve a non-coding function (e.g., promoter, origin of replication,ribosome-binding site, etc.) as well.

A first sequence is an “antisense” with respect to a second sequence ifa polynucleotide whose sequence is the first sequence specificallyhybridizes with a polynucleotide whose sequence is the second sequence.

Terms used to describe sequence relationships between two or morenucleotide sequences or amino acid sequences include “referencesequence,” “selected from,” “comparison window,” “identical,”“percentage of sequence identity,” “substantially identical,”“complementary,” and “substantially complementary.”

For sequence comparison of nucleic acid sequences, typically onesequence acts as a reference sequence, to which test sequences arecompared. When using a sequence comparison algorithm, test and referencesequences are entered into a computer, subsequence coordinates aredesignated, if necessary, and sequence algorithm program parameters aredesignated. Default program parameters are used. Methods of alignment ofsequences for comparison are well known in the art. Optimal alignment ofsequences for comparison can be conducted, e.g., by the local homologyalgorithm of Smith and Waterman, Adv. Appl. Math. 2:482, 1981, by thehomology alignment algorithm of Needleman and Wunsch, J. Mol. Biol.48:443, 1970, by the search for similarity method of Pearson & Lipman,Proc. Nat'l. Acad. Sci. USA 85:2444, 1988, by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by manual alignment and visualinspection (see, e.g., Current Protocols in Molecular Biology (Ausubelet al., eds 1995 supplement)).

One example of a useful algorithm is PILEUP. PILEUP uses asimplification of the progressive alignment method of Feng & Doolittle,J. Mol. Evol. 35:351-360, 1987. The method used is similar to the methoddescribed by Higgins & Sharp, CABIOS 5:151-153, 1989. Using PILEUP, areference sequence is compared to other test sequences to determine thepercent sequence identity relationship using the following parameters:default gap weight (3.00), default gap length weight (0.10), andweighted end gaps. PILEUP can be obtained from the GCG sequence analysissoftware package, e.g., version 7.0 (Devereaux et al., Nuc. Acids Res.12:387-395, 1984.

Another example of algorithms that are suitable for determining percentsequence identity and sequence similarity are the BLAST and the BLAST2.0 algorithm, which are described in Altschul et al., J. Mol. Biol.215:403-410, 1990 and Altschul et al., Nucleic Acids Res. 25:3389-3402,1977. Software for performing BLAST analyses is publicly availablethrough the National Center for Biotechnology Information(ncbi.nlm.nih.gov). The BLASTN program (for nucleotide sequences) usesas defaults a word length (W) of 11, alignments (B) of 50, expectation(E) of 10, M=5, N=−4, and a comparison of both strands. The BLASTPprogram (for amino acid sequences) uses as defaults a word length (W) of3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915, 1989). Anoligonucleotide is a linear polynucleotide sequence of up to about 100nucleotide bases in length.

ClustalW is a program that aligns three or more sequences in acomputationally efficient manner Aligning multiple sequences highlightsareas of similarity which may be associated with specific features thathave been more highly conserved than other regions. Thus, this programcan classify sequences for phylogenetic analysis, which aims to modelthe substitutions that have occurred over evolution and derive theevolutionary relationships between sequences. The ClustalW multiplesequence alignment web form is available on the internet from EMBL-EBI(ebi.ac.uk/Tools/msa/clustalw2/), see also Larkin et al., Bioinformatics23(21): 2947-2948, 2007.

A polynucleotide or nucleic acid sequence refers to a polymeric form ofnucleotide at least 10 bases in length. A recombinant polynucleotideincludes a polynucleotide that is not immediately contiguous with bothof the coding sequences with which it is immediately contiguous (one onthe 5′ end and one on the 3′ end) in the naturally occurring genome ofthe organism from which it is derived. The term therefore includes, forexample, a recombinant DNA which is incorporated into a vector; into anautonomously replicating plasmid or virus; or into the genomic DNA of aprokaryote or eukaryote, or which exists as a separate molecule (e.g., acDNA) independent of other sequences. The nucleotides can beribonucleotides, deoxyribonucleotides, or modified forms of eithernucleotide. The term includes single- and double-stranded forms of DNA.

Pharmaceutically acceptable carriers: The pharmaceutically acceptablecarriers of use are conventional. Remington's Pharmaceutical Sciences,by E. W. Martin, Mack Publishing Co., Easton, Pa., 19th Edition, 1995,describes compositions and formulations suitable for pharmaceuticaldelivery of the antibodies herein disclosed.

In general, the nature of the carrier will depend on the particular modeof administration being employed. For instance, parenteral formulationsusually comprise injectable fluids that include pharmaceutically andphysiologically acceptable fluids, which include, but are not limitedto, water, physiological saline, balanced salt solutions, aqueousdextrose, glycerol or the like as a vehicle. For solid compositions(e.g., powder, pill, tablet, or capsule forms), conventional non-toxicsolid carriers can include, for example, pharmaceutical grades ofmannitol, lactose, starch, or magnesium stearate. In addition tobiologically neutral carriers, pharmaceutical compositions to beadministered can contain minor amounts of non-toxic auxiliarysubstances, such as wetting or emulsifying agents, preservatives, and pHbuffering agents and the like, for example sodium acetate or sorbitanmonolaurate.

Pharmaceutical agent: A chemical compound or composition capable ofinducing a desired therapeutic or prophylactic effect when properlyadministered to a subject or a cell. In some examples a pharmaceuticalagent includes one or more of the disclosed antibodies.

Polypeptide: Any chain of amino acids, regardless of length orpost-translational modification (e.g., glycosylation orphosphorylation). In one embodiment, the polypeptide is NoV polypeptide,such as a capsid polypeptide. In one embodiment, the polypeptide is adisclosed antibody or a fragment thereof. A “residue” refers to an aminoacid or amino acid mimetic incorporated in a polypeptide by an amidebond or amide bond mimetic. A polypeptide has an amino terminal(N-terminal) end and a carboxy terminal end. Conservative amino acidsubstitution tables providing functionally similar amino acids are wellknown to one of ordinary skill in the art. The following six groups areexamples of amino acids that are considered to be conservativesubstitutions for one another:

-   -   1) Alanine (A), Serine (S), Threonine (T);    -   2) Aspartic acid (D), Glutamic acid (E);    -   3) Asparagine (N), Glutamine (Q);    -   4) Arginine (R), Lysine (K);    -   5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and    -   6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

Purified: The term purified does not require absolute purity; rather, itis intended as a relative term. Thus, for example, a purified peptidepreparation is one in which the peptide or protein (such as an antibody)is more enriched than the peptide or protein is in its naturalenvironment within a cell. In one embodiment, a preparation is purifiedsuch that the protein or peptide represents at least 50%, 60%, 70%, 80%,90%, 95%, 96%, 97%, 98%, or 99% of the total peptide or protein contentof the preparation.

Recombinant: A recombinant nucleic acid is one that has a sequence thatis not naturally occurring or has a sequence that is made by anartificial combination of two otherwise separated segments of sequence.This artificial combination is often accomplished by chemical synthesisor, more commonly, by the artificial manipulation of isolated segmentsof nucleic acids, e.g., by genetic engineering techniques.

Sequence identity: The similarity between amino acid sequences isexpressed in terms of the similarity between the sequences, otherwisereferred to as sequence identity. Sequence identity is frequentlymeasured in terms of percentage identity (or similarity or homology);the higher the percentage, the more similar the two sequences are.Homologs or variants of a polypeptide will possess a relatively highdegree of sequence identity when aligned using standard methods.

Methods of alignment of polypeptide sequences for comparison are wellknown in the art. Various programs and alignment algorithms aredescribed in: Smith and Waterman, Adv. Appl. Math. 2:482, 1981;Needleman and Wunsch, J. Mol. Biol. 48:443, 1970; Pearson and Lipman,Proc. Natl. Acad. Sci. U.S.A. 85:2444, 1988; Higgins and Sharp, Gene73:237, 1988; Higgins and Sharp, CABIOS 5:151, 1989; Corpet et al.,Nucleic Acids Research 16:10881, 1988; and Pearson and Lipman, Proc.Natl. Acad. Sci. U.S.A. 85:2444, 1988. Altschul et al., Nature Genet.6:119, 1994, presents a detailed consideration of sequence alignmentmethods and homology calculations. The NCBI Basic Local Alignment SearchTool (BLAST) (Altschul et al., J. Mol. Biol. 215:403, 1990) is availablefrom several sources, including the National Center for BiotechnologyInformation (NCBI, Bethesda, Md.) and on the internet (along with adescription of how to determine sequence identity using this program).

Homologs and variants of a V_(L) or a V_(H) of an antibody, or a V_(H)Hmonoclonal antibody, that specifically binds a polypeptide are typicallycharacterized by possession of at least about 75%, for example at leastabout 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%sequence identity counted over the full length alignment with the aminoacid sequence of interest. Proteins with even greater similarity to thereference sequences will show increasing percentage identities whenassessed by this method, such as at least 80%, at least 85%, at least90%, at least 95%, at least 98%, or at least 99% sequence identity. Whenless than the entire sequence is being compared for sequence identity,homologs and variants will typically possess at least 80% sequenceidentity over short windows of 10-20 amino acids, and may possesssequence identities of at least 85% or at least 90% or 95% depending ontheir similarity to the reference sequence. One of skill in the art willappreciate that these sequence identity ranges are provided for guidanceonly; it is entirely possible that strongly significant homologs couldbe obtained that fall outside of the ranges provided.

Nucleic acids that “selectively hybridize” or “selectively bind” do sounder moderately or highly stringent conditions that excludesnon-related nucleotide sequences. In nucleic acid hybridizationreactions, the conditions used to achieve a particular level ofstringency will vary, depending on the nature of the nucleic acids beinghybridized. For example, the length, degree of complementarity,nucleotide sequence composition (for example, GC v. AT content), andnucleic acid type (for example, RNA versus DNA) of the hybridizingregions of the nucleic acids can be considered in selectinghybridization conditions. An additional consideration is whether one ofthe nucleic acids is immobilized, for example, on a filter.

A specific example of progressively higher stringency conditions is asfollows: 2×SSC/0.1% SDS at about room temperature (hybridizationconditions); 0.2×SSC/0.1% SDS at about room temperature (low stringencyconditions); 0.2×SSC/0.1% SDS at about 42° C. (moderate stringencyconditions); and 0.1×SSC at about 68° C. (high stringency conditions).One of skill in the art can readily determine variations on theseconditions (e.g., Molecular Cloning: A Laboratory Manual, 2nd ed., vol.1-3, ed. Sambrook et al., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N Y, 1989). Washing can be carried out using only one ofthese conditions, e.g., high stringency conditions, or each of theconditions can be used, e.g., for 10-15 minutes each, in the orderlisted above, repeating any or all of the steps listed. However, asmentioned above, optimal conditions will vary, depending on theparticular hybridization reaction involved, and can be determinedempirically.

Specifically bind: When referring to an antibody, refers to a bindingreaction which determines the presence of a target protein, peptide, orpolysaccharide in the presence of a heterogeneous population of proteinsand other biologics. Thus, under designated conditions, an antibodybinds preferentially to a particular target protein, peptide orpolysaccharide (such as an antigen present on the surface of a pathogen,for example VP1 or any other NoV polypeptide) and do not bind in asignificant amount to other proteins or polysaccharides present in thesample or subject. Specific binding can be determined by methods knownin the art. With reference to an antibody antigen complex, specificbinding of the antigen and antibody has a K_(d) of less than about 10⁻⁷Molar, such as less than about 10⁻⁷ Molar, 10⁻⁸ Molar, 10⁻⁹, or evenless than about 10⁻¹⁰ Molar for a primate antibody. The antigenspecificity and affinity of V_(H)H from immune libraries Kinetickon andkoff rate constants in the ranges of 10⁵ to 10⁶ M⁻¹s⁻¹ and 10⁻² to 10⁻⁴s⁻¹, respectively, are routinely obtained so that low nanomolar or evenpicomolar equilibrium dissociation constants are obtained. Such affinityparameters are excellent for most applications (Muyldermans, Annu. Rev.Biochem. 2013. 82:775-97, 2013).

Therapeutic agent: Used in a generic sense, it includes treating agents,prophylactic agents, and replacement agents.

Therapeutically effective amount or effective amount: A quantity of aspecific substance, such as a disclosed antibody, sufficient to achievea desired effect in a subject being treated. For instance, this can bethe amount necessary to inhibit NoV replication or NV replication, or totreat an infection with the virus. In several embodiments, atherapeutically effective amount is the amount necessary to reduce asign or symptom of the infection, and/or to decrease viral titer in asubject. When administered to a subject, a dosage will generally be usedthat will achieve target tissue concentrations that has been shown toachieve a desired in vitro effect.

Vector: A nucleic acid molecule as introduced into a host cell, therebyproducing a transformed host cell. A vector may include nucleic acidsequences that permit it to replicate in a host cell, such as an originof replication. A vector may also include one or more selectable markergenes and other genetic elements known in the art.

V_(H)H: The variable domain of a camelid heavy chain antibody is calledV_(H)H, and is comprised of only one polypeptide chain. A naturallyoccurring V_(H)H has a molecular weight of approximately 15 kDa. Thereis no Fc region in a V_(H)H. DNA encoding a V_(H)H can be modified bygenetic engineering to yield a small recombinant protein having highaffinity for a target, resulting in a low molecular weightantibody-derived protein known as a camelid “nanoantibody” or“nanobody”. See U.S. Pat. No. 5,759,808 issued Jun. 2, 1998; see alsoStijlemans et al., J Biol Chem 279: 1256-1261, 2004; Dumoulin et al.,Nature 424: 783-788, 2003; Pleschberger et al., Bioconjugate Chem 14:440-448, 2003; Cortez-Retamozo et al. Int J Cancer 89: 456-62, 2002; andLauwereys et al., EMBO J 17: 3512-3520, 1998.

A V_(H)H includes in an N- to C-direction, the following structuraldomains: N-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-C, wherein FR denotes aframework region amino acid sequence and CDR denotes a complementarydetermining region amino acid sequence (see, e.g., Kabat et al.,Sequences of Proteins of Immunological Interest, U.S. Department ofHealth and Human Services, 1991). In specific non-limiting examples, theCDR3 comprises a llama CDR3 V_(H)H domain amino acid sequence; andwherein the antibody binds to Norovirus (NoV). V_(H)H monoclonalantibodies have only a heavy chain, and thus include only one CDR1, CDR2and CDR3. Generally, the CDR3 is primarily responsible for antigenspecificity.

Virus: Microscopic infectious organism that reproduces inside livingcells. A virus consists essentially of a core of a single nucleic acidsurrounded by a protein coat, and has the ability to replicate onlyinside a living cell. “Viral replication” is the production ofadditional virus by the occurrence of at least one viral life cycle. Avirus may subvert the host cells' normal functions, causing the cell tobehave in a manner determined by the virus. For example, a viralinfection may result in a cell producing a cytokine, or responding to acytokine, when the uninfected cell does not normally do so.

The singular terms “a,” “an,” and “the” include plural referents unlesscontext clearly indicates otherwise. Similarly, the word “or” isintended to include “and” unless the context clearly indicatesotherwise. It is further to be understood that all base sizes or aminoacid sizes, and all molecular weight or molecular mass values, given fornucleic acids or polypeptides are approximate, and are provided fordescription. Although methods and materials similar or equivalent tothose described herein can be used in the practice or testing of thisdisclosure, suitable methods and materials are described below. The term“comprises” means “includes.” All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including explanations of terms, will control. Inaddition, the materials, methods, and examples are illustrative only andnot intended to be limiting.

V_(H)H Monoclonal Antibodies that Specifically Bind NoV

Isolated single domain monoclonal antibodies, V_(H)H, and antigenbinding fragments thereof that specifically bind a NoV polypeptide, andspecifically bind a NoV, are disclosed herein. In some embodiments, theantibody specifically binds a NoV Genogroup I or a NoV Genogroup IIrecombinant virus like particles (VLPs). In some embodiments, theantibody specifically binds a Norwalk virus VLP or a MD2004 virus VLP.The antibody can specifically bind a viral structural capsid protein(VP) 1. In some examples, the monoclonal antibody specifically binds aP1 or P2 subdomain of VP1. The monoclonal antibody can specifically bindthe NoV polypeptide and/or VLPs.

In some embodiments the monoclonal antibodies or antigen bindingfragment thereof specifically bind a NoV VPLs with an equilibriumconstant (K_(d)) of 1 nM or less. In several embodiments, the antibodiesand antigen binding fragments bind the NoV polypeptide, such as aNorwalk virus polypeptide, or an MD2004 virus polypeptide, with abinding affinity of 1×10⁻⁹ M, at least about 1.5×10⁻⁹M, at least about2×10⁻⁹M, at least about 3×10⁻⁹ M, at least about 3×10⁻⁹ M, at leastabout 5×10⁻⁹M, at least about 6×10⁻⁹M, at least about 7×10⁻⁹ M, at leastabout 8×10⁻⁹M, at least about 9×10⁻⁹M, or at least about 1×10⁻¹⁰ M.

In some embodiments, the antibody is neutralizing. In furtherembodiments the antibody is broadly neutralizing, such as for all ormany Genogroup I or Genogroup II NoV. In other embodiments, the antibodyinhibits the binding of NoV VLPs to HBGA synthetic carbohydrates, forexample H1 or H3 type HBGA, in a dose dependent manner. In otherembodiments the antibody inhibits the binding of NoV-VLPs to pig gastricmucin or saliva, in a dose dependent manner. In additional embodiments,the antibody is inhibitory in a hemagglutination assay.

The monoclonal antibody can be of any isotype. The single domainmonoclonal antibody can be, for example, an IgG antibody. The class ofan antibody can be switched with another. In one aspect, a nucleic acidmolecule encoding the V_(H)H is isolated using methods well-known in theart, such that it does not include any nucleic acid sequences encodingthe constant region. The nucleic acid molecule encoding the V_(H)H canbe operatively linked to a nucleic acid sequence encoding a C_(H) from adifferent class of immunoglobulin molecule. This can be achieved using avector or nucleic acid molecule that comprises a C_(H) chain, as knownin the art. For example, an antibody can be class switched to an IgG.Class switching can be used to convert one IgG subclass to another, suchas, but not limited to, from IgG1 to IgG2 or IgG3.

The monoclonal antibodies disclosed herein can be llama antibodies, andcan include a llama framework region. In some embodiments, theantibodies are humanized, and thus include one or more human frameworkregions. Exemplary framework regions are disclosed, for example, in PCTPublication No. WO 2011/038290 and Published U.S. Patent Application No.2012/0244166A1, which are incorporated by reference herein. In someembodiments, the antibodies disclosed herein are chimeric antibodies. Insome embodiments, the antibodies include llama and human regions.

In some embodiments, the monoclonal antibody includes only a heavy chainvariable (V_(H)H) domain, in the absence of a light chain domain. EachV_(H) is composed of three CDRs and four FWRs, arranged fromamino-terminus to carboxy-terminus in the following order: FWR1, CDR1,FWR2, CDR2, FWR3, CDR3, and FWR4. In humans, the heavy chain constantregion is comprised of three domains, C_(H)1, C_(H)2 and C_(H)3, whilellamas do not have a C_(H)1 domain.

CDRs and FWRs may be defined according to Kabat (Sequences of Proteinsof Immunological Interest (National Institutes of Health, Bethesda, Md.,1987 and 1991) Amino acid numbering of antibodies or antigen bindingfragments is also according to that of Kabat. Each CDR can include aminoacid residues from a complementarity determining region as defined byKabat (i.e. about residues 31-35 (CDR-H1), 50-65 (CDR-H2) and 95-102(CDR-H3) in the heavy chain variable domain (SEQ ID NO: 2). However, insome antibodies the CDRs include those residues from a hypervariableloop (i.e. about residues 26-32 (CDR-H1), 53-55 (CDR-H2) and 96-101(CDR-H3) in the heavy chain variable domain (SEQ ID NO: 2); Chothia andLesk J. Mol. Biol. 196:901-917 (1987)). In some instances, acomplementarity determining region can include amino acids from both aCDR region defined according to Kabat and a hypervariable loop. In awild type antibody, each variable domain typically has four FWRsidentified as FWR1, FWR2, FWR3 and FWR4. If the CDRs are definedaccording to Kabat, the heavy chain FWR residues are positioned about atresidues 1-30 (HCFR1), 36-49 (HCFR2), 66-94 (HCFR3), and 103-113 (HCFR4)of SEQ ID NO: 2. If the CDRs comprise amino acid residues fromhypervariable loops, the heavy chain FWR residues are positioned aboutat residues 1-25 (HCFR1), 33-52 (HCFR2), 56-95 (HCFR3), and 102-113(HCFR4) in the heavy chain (SEQ ID NO:2). In some instances, when theCDR comprises amino acids from both a CDR as defined by Kabat and thoseof a hypervariable loop, the FWR residues are adjusted accordingly.

Thus, in some embodiments, the monoclonal antibody includes one or moreheavy chain CDRs from the variable domains shown in FIG. 8, as definedby the Kabat, Chothia or IMGT numbering system.

The monoclonal antibodies disclosed herein can specifically bind NoVVLPs, such as a Genogroup I or Genogroup II NoV VLPs. NoVs arenonenveloped ˜38 nm icosahedral viruses with an approximately 7.5 kbsingle-stranded, positive-sense RNA genome that encodes three openreading frames (ORFs). ORF1 encodes RNA-dependent RNA polymerase, whileORFs 2 and 3 encode the major (VP1) and minor (VP2) capsid proteins,respectively. The VP1 is structurally divided into the shell domain (S)that forms the internal structural core of the particle and theprotruding domain (P) that is exposed on the outer surface (Prasad etal., Science 286:287-90, 1999). The P domain is further subdivided intothe P1 subdomain (residues 226 to 278 and 406 to 520) and the P2subdomain (residues 279 to 405) (Prasad et al., J Virol 68:5117-25,1999). P2 represents the most exposed surface of the viral particle andis involved in interactions with both neutralizing antibodies and HBGAoligosaccharides (Cao et al., J Virol 81:5949-57, 2007; Chen et al.,Proc Natl Acad Sci USA 103:8048-53, 2006; Lochridge et al., J Gen Virol86:2799-806, 2005).

NoVs are divided into five distinct genogroups (GI-GV) based on VP1sequence similarity. Different types within each Genogroup are separatedfrom the Genogrop by a decimal point. Virus strains from GI and GII areresponsible for most human infections, and these genogroups are furthersubdivided into more than 30 different genotypes (Kroneman et al., ArchVirol 158(10): 2059-68, 2013.). Although human NoV GII.4 strains are nowrecognized as the predominant genotype, the GI.1 Norwalk virus (NV) hasbeen studied most extensively because of its historical precedence(Kapikian J Infect Dis 181 Suppl 2:S295-302, 2000). Early humanchallenge studies with NV provided evidence for short-term, but notlong-term (>2 years), homologous immunity following infection with NVand showed also the absence of heterotypic immunity whencross-challenged with the GII.1 Hawaii virus (Wyatt et al., J Infect Dis129:709-14, 1974). Later human challenge studies showed an associationbetween HBGA secretor status and susceptibility to NV infection(Harrington et al., J Virol 76:12335-43, 2002; Hutson et al., J InfectDis 185:1335-7, 2002; Lindesmith et al., Nat Med 9(5):548-53, 2003;Marionneau et al., Gastroenterology 122:1967-77, 2002). The elucidationof human NoV virion structure is based largely on the X-raycrystallographic analysis of NoV rVLPs (Prasad et al., Science286:287-90, 1999; Prasad et al., J Virol 68:5117-25, 1994); these VLPsare a promising NoV vaccine candidate (Atmar et al., N Engl J Med365:2178-87, 2011).

Examples of NoVs include, but are not limited to, NV (NV, see, forexample, GENBANK® Accession No. M87661, NP_056821), P7-587 virus (see,for example, GENBANK® Accession No. FJ384783), Southampton virus (SHV,see, for example, GENBANK® Accession No. L07418), Desert Shield virus(DSV, see, for example, GENBANK® Accession No. U04469), Hesse virus(HSV, see, for example, GENBANK® Accession No. AF093797), Chiba virus(CHV, see, for example, GENBANK® Accession No. AB042808), Hawaii virus(HV, see, for example, GENBANK® Accession No. U07611), Snow Mountainvirus (SMV, see, for example, GENBANK® Accession No. U70059), Torontovirus (TV, see for example, GENBANK® Accession No. U02030), Bristolvirus (BV, see for example, GENBANK® Accession No. X76716), Jena virus(JV, see, for example, GENBANK® Accession No. AJ011099), MD145-12 virus(MD145-12, see, for example, GENBANK® Accession No. AY032605), Aichivirus (AV124-89, see, for example, GENBANK® Accession No. AB031013),Camberwell (CV, see, for example, GENBANK® Accession No. AF145896),Lordsdale virus (LV, see, for example, GENBANK® Accession No. X86557),Grimsby virus (GrV, see, for example, GENBANK® Accession No. AJ004864),Mexico virus (MXV, see, for example, GENBANK® Accession No. U22498),Boxer (see, for example, GENBANK® Accession No. AF538679), C59 (see, forexample, GENBANK® Accession No. AF435807), VA98115 (see, for example,GENBANK® Accession No. AY038598), BUDS (see, for example, GENBANK®Accession No. AY660568), MOH (see, for example, GENBANK® Accession No.AF397156), Parris Island (PiV; see, for example, GENBANK® Accession No.AY652979), VA98387 (see, for example, GENBANK® Accession No. AY038600),VA97207 (see, for example, GENBANK® Accession No. AY038599), andOperation Iraqi Freedom (see, for example, OIF, GENBANK® Accession No.AY675554).

In some embodiments, the monoclonal antibody specifically binds aGenogroup I NoVs, which include, but are not limited to, Norwalk virus.In additional embodiments, the antibody specifically binds Genogroup IINoVs, which include, but are not limited to, MD2004 virus.

In some embodiments, the monoclonal antibody, or antigen bindingfragment thereof specifically binds a Genogroup II NoV polypeptide, andincludes a CDR3, wherein the CDR3 includes the amino acid sequence setforth as one of:

-   -   a) amino acids 96-109 of SEQ ID NO: 1;    -   b) amino acids 96-109 of SEQ ID NO: 2;    -   c) amino acids 96-109 of SEQ ID NO: 3;    -   d) amino acids 96-109 of SEQ ID NO: 4;    -   e) amino acids 97-110 of SEQ ID NO: 5;    -   f) amino acids 97-111 of SEQ ID NO: 6;    -   g)amino acids 97-111 of SEQ ID NO: 7;    -   h) amino acids 97-111 of SEQ ID NO: 8;    -   i) amino acids 96-112 of SEQ ID NO: 9;    -   j) amino acids 96-112 of SEQ ID NO: 10;    -   k) amino acids 96-114 of SEQ ID NO: 11;    -   l) amino acids 96-112 of SEQ ID NO: 12;    -   m) amino acids 97-113 of SEQ ID NO: 13;    -   n) amino acids 97-113 of SEQ ID NO: 14;    -   o) amino acids 97-114 of SEQ ID NO: 15;    -   p) amino acids 96-107 of SEQ ID NO: 16;    -   q) amino acids 97-113 of SEQ ID NO: 17;    -   r) amino acids 97-114 of SEQ ID NO: 18;    -   s) amino acids 97-113 of SEQ ID NO: 19;    -   t) amino acids 96-111 of SEQ ID NO: 20; or    -   u) amino acids 96-102 of SEQ ID NO: 21.        The monoclonal antibody can be a V_(H)H monoclonal antibody.

In other embodiments, the monoclonal antibody, or antigen bindingfragment thereof, specifically binds a Genogroup I NoV polypeptide, andincludes a CDR3, wherein the CDR3 includes the amino acid sequence setforth as one of:

-   -   a) amino acids 96-110 of SEQ ID NO: 22;    -   b) amino acids 95-104 of SEQ ID NO: 23;    -   c) amino acids 96-107 of SEQ ID NO: 24;    -   d) amino acids 100-110 of SEQ ID NO: 25;    -   e) amino acids 96-117 of SEQ ID NO: 26;    -   f) amino acids 97-108 of SEQ ID NO: 27;    -   g) amino acids 96-112 of SEQ ID NO: 28;    -   h) amino acids 97-115 of SEQ ID NO: 29; or    -   i) amino acids 97-121 of SEQ ID NO: 30.        The monoclonal antibody can be a V_(H)H monoclonal antibody.

In some embodiments, the monoclonal antibody, or antigen bindingfragment thereof, specifically binds a Genogroup II NoV polypeptide, andincludes:

-   -   a) a CDR1 comprising amino acids 26-33 of SEQ ID NO: 1, a CDR2        comprising amino acids 51-57 of SEQ ID NO: 1, and/or a CDR3        comprising amino acids 96-109 of SEQ ID NO: 1;    -   b) a CDR1 comprising amino acids 26-33 of SEQ ID NO: 2, a CDR2        comprising amino acids 51-57 of SEQ ID NO: 2, and/or a CDR3        comprising amino acids 96-109 of SEQ ID NO: 2;    -   c) a CDR1 comprising amino acids 26-33 of SEQ ID NO: 3, a CDR2        comprising amino acids 51-57 of SEQ ID NO: 3, and/or a CDR3        comprising amino acids 96-109 of SEQ ID NO: 3;    -   d) a CDR1 comprising amino acids 26-33 of SEQ ID NO: 4, a CDR2        comprising amino acids 51-57 of SEQ ID NO: 4, and/or a CDR3        comprising amino acids 96-109 of SEQ ID NO: 4;    -   e) a CDR1 comprising amino acids 26-33 of SEQ ID NO: 5, a CDR2        comprising amino acids 51-58 of SEQ ID NO: 5, and/or a CDR3        comprising amino acids 97-110 of SEQ ID NO: 5;    -   f) a CDR1 comprising amino acids 26-33 of SEQ ID NO: 6, a CDR2        comprising amino acids 51-58 of SEQ ID NO: 6, and/or a CDR5        comprising amino acids 97-111 of SEQ ID NO: 6;    -   g) a CDR1 comprising amino acids 26-33 of SEQ ID NO: 7, a CDR2        comprising amino acids 51-58 of SEQ ID NO: 7, and/or a CDR3        comprising amino acids 97-111 of SEQ ID NO: 7;    -   h) a CDR1 comprising amino acids 26-33 of SEQ ID NO: 8, a CDR2        comprising amino acids 51-58 of SEQ ID NO: 8, and/or a CDR3        comprising amino acids 97-111 of SEQ ID NO: 8;    -   i) a CDR1 comprising amino acids 26-33 of SEQ ID NO: 9, a CDR2        comprising amino acids 51-57 of SEQ ID NO: 9, and/or a CDR3        comprising amino acids 96-112 of SEQ ID NO: 9;    -   j) a CDR1 comprising amino acids 26-33 of SEQ ID NO: 10, a CDR2        comprising amino acids 51-57 of SEQ ID NO: 10, and/or a CDR3        comprising amino acids 96-112 of SEQ ID NO: 10;    -   k) a CDR1 comprising amino acids 26-33 of SEQ ID NO: 11, a CDR2        comprising amino acids 51-57, and/or a CDR3 comprising amino        acids 96-114 of SEQ ID NO: 11;    -   l) a CDR1 comprising amino acids 26-33 of SEQ ID NO: 12, a CDR2        comprising amino acids 51-57 of SEQ ID NO: 12, and/or a CDR3        comprising amino acids 96-112 of SEQ ID NO: 12;    -   m) a CDR1 comprising amino acids 26-33 of SEQ ID NO: 13, a CDR2        comprising amino acids 51-58 of SEQ ID NO: 13, and/or a CDR3        comprising amino acids 97-113 of SEQ ID NO: 13;    -   n) a CDR1 comprising amino acids 26-33 of SEQ ID NO: 14, a CDR2        comprising amino acids 51-58 of SEQ ID NO: 14, and/or a CDR3        comprising amino acids 97-113 of SEQ ID NO: 14;    -   o) a CDR1 comprising amino acids 26-33 of SEQ ID NO: 15, a CDR2        comprising amino acids 51-58 of SEQ ID NO: 15, and/or a CDR3        comprising amino acids 97-114 of SEQ ID NO: 15;    -   p) a CDR1 comprising amino acids 26-33 of SEQ ID NO: 16, a CDR2        comprising amino acids 51-57 of SEQ ID NO: 16, and/or a CDR3        comprising amino acids 96-107 of SEQ ID NO: 16;    -   q) a CDR1 comprising amino acids 26-33 of SEQ ID NO: 17, a CDR2        comprising amino acids 51-58 of SEQ ID NO: 17, and/or a CDR3        comprising amino acids 97-113 of SEQ ID NO: 17;    -   r) a CDR1 comprising amino acids 26-33 of SEQ ID NO: 18, a CDR2        comprising amino acids 51-58 of SEQ ID NO: 18, and/or a CDR3        comprising amino acids 97-114 of SEQ ID NO: 18;    -   s) a CDR1 comprising amino acids 26-33 of SEQ ID NO: 19, a CDR2        comprising amino acids 51-58 of SEQ ID NO: 19, and/or a CDR3        comprising amino acids 97-113 of SEQ ID NO: 19;    -   t) a CDR1 comprising amino acids 26-33 of SEQ ID NO: 20, a CDR2        comprising amino acids 51-57 of SEQ ID NO:20, and/or a CDR3        comprising amino acids 96-111 of SEQ ID NO: 20; or    -   u) a CDR1 comprising amino acids 26-33 of SEQ ID NO: 21, a CDR2        comprising amino acids 51-57 of SEQ ID NO:21, and/or a CDR3        comprising amino acids 96-102 of SEQ ID NO: 21.        The monoclonal antibody can be a V_(H)H monoclonal antibody.

In other embodiments, the monoclonal antibody, or antigen bindingfragment thereof, specifically binds a Genogroup II NoV polypeptide, andincludes:

-   -   a) a CDR1 comprising amino acids 26-33 of SEQ ID NO: 22, a CDR2        comprising amino acids 51-57 of SEQ ID NO:22, and/or a CDR3        comprising amino acids 96-110 of SEQ ID NO: 22;    -   b) a CDR1 comprising amino acids 26-33 of SEQ ID NO: 23, a CDR2        comprising amino acids 50-56 of SEQ ID NO:23, and/or a CDR3        comprising amino acids 95-104 of SEQ ID NO: 23;    -   c) a CDR1 comprising amino acids 26-33 of SEQ ID NO: 24, a CDR2        comprising amino acids 51-57 of SEQ ID NO:24, and/or a CDR3        comprising amino acids 96-107 of SEQ ID NO: 24;    -   d) a CDR1 comprising amino acids 26-33 of SEQ ID NO: 25, a CDR2        comprising amino acids 51-57 of SEQ ID NO: 25, and/or a CDR3        comprising amino acids 100-110 of SEQ ID NO: 25;    -   e) a CDR1 comprising amino acids 26-33 of SEQ ID NO: 26, a CDR2        comprising amino acids 50-57 of SEQ ID NO:26, and/or a CDR3        comprising amino acids 96-117 of SEQ ID NO: 26;    -   f) a CDR1 comprising amino acids 26-33 of SEQ ID NO: 27, a CDR2        comprising amino acids 51-58 of SEQ ID NO:27, and/or a CDR3        comprising amino acids 97-108 of SEQ ID NO: 27;    -   g) a CDR1 comprising amino acids 26-33 of SEQ ID NO: 28, a CDR2        comprising amino acids 51-57 of SEQ ID NO:28, and/or a CDR3        comprising amino acids 96-112 of SEQ ID NO: 28;    -   h) a CDR1 comprising amino acids 26-33 of SEQ ID NO: 29, a CDR2        comprising amino acids 51-58 of SEQ ID NO:29, and/or a CDR3        comprising amino acids 97-115 of SEQ ID NO: 29; or    -   i) a CDR1 comprising amino acids 26-33 of SEQ ID NO: 30, a CDR2        comprising amino acids 51-58 of SEQ ID NO:30, and/or a CDR3        comprising amino acids 97-121 of SEQ ID NO: 30.        The monoclonal antibody can be a V_(H)H monoclonal antibody.

In additional embodiments, the monoclonal antibody, or antigen bindingfragment thereof, specifically binds a Genogroup II NoV, and includes:

-   -   a) a CDR1 comprising amino acids 26-33 of SEQ ID NO: 1, a CDR2        comprising amino acids 51-57 of SEQ ID NO: 1, and a CDR3        comprising amino acids 96-109 of SEQ ID NO: 1;    -   b) a CDR1 comprising amino acids 26-33 of SEQ ID NO: 2, a CDR2        comprising amino acids 51-57 of SEQ ID NO: 2, and a CDR3        comprising amino acids 96-109 of SEQ ID NO: 2;    -   c) a CDR1 comprising amino acids 26-33 of SEQ ID NO: 3, a CDR2        comprising amino acids 51-57 of SEQ ID NO: 3, and a CDR3        comprising amino acids 96-109 of SEQ ID NO: 3;    -   d) a CDR1 comprising amino acids 26-33 of SEQ ID NO: 4, a CDR2        comprising amino acids 51-57 of SEQ ID NO: 4, and a CDR3        comprising amino acids 96-109 of SEQ ID NO: 4;    -   e) a CDR1 comprising amino acids 26-33 of SEQ ID NO: 5, a CDR2        comprising amino acids 51-58 of SEQ ID NO: 5, and a CDR3        comprising amino acids 97-110 of SEQ ID NO: 5;    -   f) a CDR1 comprising amino acids 26-33 of SEQ ID NO: 6, a CDR2        comprising amino acids 51-58 of SEQ ID NO: 6, and a CDR5        comprising amino acids 97-111 of SEQ ID NO: 6;    -   g) a CDR1 comprising amino acids 26-33 of SEQ ID NO: 7, a CDR2        comprising amino acids 51-58 of SEQ ID NO: 7, and a CDR3        comprising amino acids 97-111 of SEQ ID NO: 7;    -   h) a CDR1 comprising amino acids 26-33 of SEQ ID NO: 8, a CDR2        comprising amino acids 51-58 of SEQ ID NO: 8, and a CDR3        comprising amino acids 97-111 of SEQ ID NO: 8;    -   i) a CDR1 comprising amino acids 26-33 of SEQ ID NO: 9, a CDR2        comprising amino acids 51-57 of SEQ ID NO: 9, and a CDR3        comprising amino acids 96-112 of SEQ ID NO: 9;    -   j) a CDR1 comprising amino acids 26-33 of SEQ ID NO: 10, a CDR2        comprising amino acids 51-57 of SEQ ID NO: 10, and a CDR3        comprising amino acids 96-112 of SEQ ID NO: 10;    -   k) a CDR1 comprising amino acids 26-33 of SEQ ID NO: 11, a CDR2        comprising amino acids 51-57, and a CDR3 comprising amino acids        96-114 of SEQ ID NO: 11;    -   l) a CDR1 comprising amino acids 26-33 of SEQ ID NO: 12, a CDR2        comprising amino acids 51-57 of SEQ ID NO: 12, and a CDR3        comprising amino acids 96-112 of SEQ ID NO: 12;    -   m) a CDR1 comprising amino acids 26-33 of SEQ ID NO: 13, a CDR2        comprising amino acids 51-58 of SEQ ID NO: 13, and a CDR3        comprising amino acids 97-113 of SEQ ID NO: 13;    -   n) a CDR1 comprising amino acids 26-33 of SEQ ID NO: 14, a CDR2        comprising amino acids 51-58 of SEQ ID NO: 14, and a CDR3        comprising amino acids 97-113 of SEQ ID NO: 14;    -   o) a CDR1 comprising amino acids 26-33 of SEQ ID NO: 15, a CDR2        comprising amino acids 51-58 of SEQ ID NO: 15, and a CDR3        comprising amino acids 97-114 of SEQ ID NO: 15;    -   p) a CDR1 comprising amino acids 26-33 of SEQ ID NO: 16, a CDR2        comprising amino acids 51-57 of SEQ ID NO: 16, and a CDR3        comprising amino acids 96-107 of SEQ ID NO: 16;    -   q) a CDR1 comprising amino acids 26-33 of SEQ ID NO: 17, a CDR2        comprising amino acids 51-58 of SEQ ID NO: 17, and a CDR3        comprising amino acids 97-113 of SEQ ID NO: 17;    -   r) a CDR1 comprising amino acids 26-33 of SEQ ID NO: 18, a CDR2        comprising amino acids 51-58 of SEQ ID NO: 18, and a CDR3        comprising amino acids 97-114 of SEQ ID NO: 18;    -   s) a CDR1 comprising amino acids 26-33 of SEQ ID NO: 19, a CDR2        comprising amino acids 51-58 of SEQ ID NO: 19, and a CDR3        comprising amino acids 97-113 of SEQ ID NO: 19;    -   t) a CDR1 comprising amino acids 26-33 of SEQ ID NO: 20, a CDR2        comprising amino acids 51-57 of SEQ ID NO:20, and a CDR3        comprising amino acids 96-111 of SEQ ID NO: 20; or    -   u) a CDR1 comprising amino acids 26-33 of SEQ ID NO: 21, a CDR2        comprising amino acids 51-57 of SEQ ID NO:21, and a CDR3        comprising amino acids 96-102 of SEQ ID NO: 21.        The monoclonal antibody can be a V_(H)H monoclonal antibody.

In yet other embodiments, the monoclonal antibody, or antigen bindingfragment thereof, specifically binds a Genogroup I NoV polypeptide, andincludes:

-   -   a) a CDR1 comprising amino acids 26-33 of SEQ ID NO: 22, a CDR2        comprising amino acids 51-57 of SEQ ID NO:22, and a CDR3        comprising amino acids 96-110 of SEQ ID NO: 22;    -   b) a CDR1 comprising amino acids 26-33 of SEQ ID NO: 23, a CDR2        comprising amino acids 50-56 of SEQ ID NO:23, and a CDR3        comprising amino acids 95-104 of SEQ ID NO: 23;    -   c) a CDR1 comprising amino acids 26-33 of SEQ ID NO: 24, a CDR2        comprising amino acids 51-57 of SEQ ID NO:24, and a CDR3        comprising amino acids 96-107 of SEQ ID NO: 24;    -   d) a CDR1 comprising amino acids 26-33 of SEQ ID NO: 25, a CDR2        comprising amino acids 51-57 of SEQ ID NO: 25, and a CDR3        comprising amino acids 100-110 of SEQ ID NO: 25;    -   e) a CDR1 comprising amino acids 26-33 of SEQ ID NO: 26, a CDR2        comprising amino acids 50-57 of SEQ ID NO:26, and a CDR3        comprising amino acids 96-117 of SEQ ID NO: 26;    -   f) a CDR1 comprising amino acids 26-33 of SEQ ID NO: 27, a CDR2        comprising amino acids 51-58 of SEQ ID NO:27, and a CDR3        comprising amino acids 97-108 of SEQ ID NO: 27;    -   g) a CDR1 comprising amino acids 26-33 of SEQ ID NO: 28, a CDR2        comprising amino acids 51-57 of SEQ ID NO:28, and a CDR3        comprising amino acids 96-112 of SEQ ID NO: 28;    -   h) a CDR1 comprising amino acids 26-33 of SEQ ID NO: 29, a CDR2        comprising amino acids 51-58 of SEQ ID NO:29, and a CDR3        comprising amino acids 97-115 of SEQ ID NO: 29; or    -   i) a CDR1 comprising amino acids 26-33 of SEQ ID NO: 30, a CDR2        comprising amino acids 51-58 of SEQ ID NO:30, and a CDR3        comprising amino acids 97-121 of SEQ ID NO: 30.        The monoclonal antibody can be a V_(H)H monoclonal antibody.

In some embodiments, the monoclonal antibody includes a heavy chainvariable domain comprising an amino acid sequence at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to one of the aminoacid sequence set forth as one of SEQ ID NOs: 1-30. Thus, the monoclonalantibody can include a heavy chain variable domain comprising an aminoacid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99% identical to one of the amino acid sequence set forth as one of SEQID NOs: 1-21, and specifically bind a Genogroup II NoV polypeptide. Inspecific non-limiting examples, the monoclonal antibody can include aheavy chain variable domain comprising an amino acid sequence at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to one ofthe amino acid sequence set forth as one of SEQ ID NOs: 1-21, andinclude a CDR3 sequence 100% identical to the CDRs sequence of SEQ IDNOs: 1-21, respectively, and specifically bind a Genogroup II NoVpolypeptide. The monoclonal antibody can include a heavy chain variabledomain comprising an amino acid sequence at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% identical to one of the amino acidsequence set forth as one of SEQ ID NOs: 22-30, and specifically bind aGenogroup I NoV polypeptide. In specific non-limiting examples, themonoclonal antibody can include a heavy chain variable domain comprisingan amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98% or 99% identical to one of the amino acid sequence set forth as oneof SEQ ID NOs: 22-30, and include a CDR3 sequence 100% identical to theCDRs sequence of SEQ ID NOs: 22-30, respectively, and specifically binda Genogroup I NoV polypeptide. These monoclonal antibodies can be V_(H)Hmonoclonal antibodies.

In additional embodiments, the heavy chain variable domain includes atmost 10, at most 9, at most 8, at most 7, at most 6, at most 5, at most4, at most 3, at most two or at most one conservative amino acidsubstitutions in an amino acid sequence set forth as one of SEQ ID NOs:1-30. In yet other embodiments, the heavy chain variable domain includesat most 10, at most 9, at most 8, at most 7, at most 6, at most 5, atmost 4, at most 3, at most two or at most one conservative amino acidsubstitutions in an amino acid sequence set forth as one of SEQ ID NOs:1-21, and specifically binds a Genogroup II NoV polypeptide. In furtherembodiments, the heavy chain variable domain includes at most 10, atmost 9, at most 8, at most 7, at most 6, at most 5, at most 4, at most3, at most two or at most one conservative amino acid substitutions inan amino acid sequence set forth as one of SEQ ID NOs: 22-30, andspecifically binds a Genogroup I NoV polypeptide. These monoclonalantibodies can be V_(H)H monoclonal antibodies.

In yet other embodiments, the monoclonal antibody includes a heavy chainvariable domain comprising or consisting of one of the amino acidsequence set forth as one of SEQ ID NOs: 1-30. The monoclonal antibodycan include a heavy chain variable domain comprising or consisting ofone of the amino acid sequences set forth as one of SEQ ID NOs: 1-21,and specifically bind a Genogroup II NoV polypeptide. The monoclonalantibody can include a heavy chain variable domain comprising orconsisting of one of the amino acid sequences set forth as one of SEQ IDNOs: 22-30, and specifically bind a Genogroup I NoV polypeptide. Thesemonoclonal antibodies can be V_(H)H monoclonal antibodies.

The monoclonal antibodies, including V_(H)H monoclonal antibodies, orantibody fragments disclosed herein can be derivatized or linked toanother molecule (such as another peptide, protein, enzyme, chromogen orantibody). In general, the antibody or portion thereof is derivatizedsuch that the binding to the NoV VLPs, for example a Norwalk virus, suchas VP1, is not affected adversely by the derivatization or labeling. Forexample, the antibody can be functionally linked, for example, bychemical coupling, genetic fusion, noncovalent association or otherwiseto one or more other molecular entities, such as another antibody (forexample, to form a bispecific antibody or to link it to a diabody), adetection agent, a pharmaceutical agent, and/or a protein or peptidethat can mediate associate of the antibody or antibody portion withanother molecule (such as a streptavidin core region or a polyhistidinetag).

One type of derivatized antibody is produced by cross-linking two ormore antibodies (of the same type or of different types, such as tocreate bispecific antibodies). Suitable crosslinkers include those thatare heterobifunctional, having two distinctly reactive groups separatedby an appropriate spacer (such asm-maleimidobenzoyl-N-hydroxysuccinimide ester) or homobifunctional (suchas disuccinimidyl suberate). Such linkers are available from PierceChemical Company (Rockford, Ill.).

A monoclonal antibody, including V_(H)H monoclonal antibody, thatspecifically binds a NoV polypeptide, such as a Genogroup I or GenogroupII NoV polypeptide, for example, a NV polypeptide, can be labeled with adetectable moiety. Useful detection agents include fluorescentcompounds, including fluorescein, fluorescein isothiocyanate, rhodamine,5-dimethylamine-1-napthalenesulfonyl chloride, phycoerythrin, lanthanidephosphors, ALEXA FLUOR® and the like. Bioluminescent markers are also ofuse, such as luciferase, green fluorescent protein (GFP), or yellowfluorescent protein. An antibody can also be labeled with enzymes thatare useful for detection, such as horseradish peroxidase,β-galactosidase, luciferase, alkaline phosphatase, glucose oxidase andthe like. When an antibody is labeled with a detectable enzyme, it canbe detected by adding additional reagents that the enzyme uses toproduce a reaction product that can be discerned. For example, when theagent horseradish peroxidase is present the addition of hydrogenperoxide and diaminobenzidine leads to a colored reaction product, whichis visually detectable. An antibody may also be labeled with biotin, anddetected through indirect measurement of avidin or streptavidin binding.It should be noted that the avidin itself can be labeled with an enzymeor a fluorescent label.

A monoclonal antibody including V_(H)H monoclonal antibody, may belabeled with a magnetic agent, such as gadolinium. Antibodies can alsobe labeled with lanthanides (such as europium and dysprosium), andmanganese. Paramagnetic particles such as superparamagnetic iron oxideare also of use as labels. An antibody may also be labeled with apredetermined polypeptide epitopes recognized by a secondary reporter(such as leucine zipper pair sequences, binding sites for secondaryantibodies, metal binding domains, epitope tags). In some embodiments,labels are attached by spacer arms of various lengths to reducepotential steric hindrance.

An antibody can also be labeled with a radiolabeled amino acid. Examplesof radiolabels include, but are not limited to, the followingradioisotopes or radionucleotides: ³H, ¹⁴C, ¹⁵ N, ³⁵S, ⁹⁰Y, ⁹⁹Tc, ¹¹¹I,¹²⁵I, ¹³¹I. The radiolabel may be used for both diagnostic andtherapeutic purposes.

Means of detecting such labels are well known to those of skill in theart. Thus, for example, radiolabels may be detected using photographicfilm or scintillation counters, fluorescent markers may be detectedusing a photodetector to detect emitted illumination. Enzymatic labelsare typically detected by providing the enzyme with a substrate anddetecting the reaction product produced by the action of the enzyme onthe substrate, and colorimetric labels are detected by simplyvisualizing the colored label.

An antibody, including V_(H)H monoclonal antibodies, can also bederivatized with a chemical group such as polyethylene glycol (PEG), amethyl or ethyl group, or a carbohydrate group. These groups may beuseful to improve the biological characteristics of the antibody, suchas to increase serum half-life or to increase tissue binding. Animmunoadhesin can be produced.

Means of detecting such labels are well known to those of skill in theart. Thus, for example, radiolabels may be detected using photographicfilm or scintillation counters, fluorescent markers may be detectedusing a photodetector to detect emitted illumination. Enzymatic labelsare typically detected by providing the enzyme with a substrate anddetecting the reaction product produced by the action of the enzyme onthe substrate, and colorimetric labels are detected by simplyvisualizing the colored label.

Polynucleotides, Expression and Production

Nucleotide sequences are provided herein that encode a monoclonalantibody, such as a V_(H)H monoclonal antibody, or an antigen bindingfragment thereof, that specifically binds a NoV polypeptide, such as aGengroup II or Genogroup I NoV polypeptide. The nucleic acid sequencecan encode a V_(H)H monoclonal antibody that specifically binds a MD2004virus polypeptide or a Norwalk virus polypeptide. The antibody canspecifically bind a polypeptide, such as VP1 or VP2. Expression vectorsare also provided for their efficient expression in cells (for example,mammalian cells, plant cells, chloroplasts insect cells, yeast, orbacteria).

Recombinant expression of an antibody generally requires construction ofan expression vector containing a polynucleotide that encodes theantibody or antibody fragment. Replicable vectors are provided includinga nucleotide sequence encoding a V_(H)H antibody molecule, and/or one ormore a heavy chain CDRs, and optionally a light chain variable domain,operably linked to a promoter. Such vectors may include the nucleotidesequence encoding the constant region of an antibody molecule (see,e.g., U.S. Pat. Nos. 5,981,216; 5,591,639; 5,658,759 and 5,122,464) andthe variable domain of the V_(H)H antibody may be cloned into such avector. Nucleic acid molecules (also referred to as polynucleotides)encoding the polypeptides provided herein (including, but not limited toV_(H)H antibodies) can readily be produced by one of skill in the art.For example, these nucleic acids can be produced using the amino acidsequences provided herein (such as the CDR sequences or variable domainsequences), and optionally sequences available in the art (such asframework sequences), and the genetic code.

V_(H)H nucleic acid sequences are set forth as SEQ ID NOs: 39-68 andinclude degenerate variants thereof. One of skill in the art can readilyuse the genetic code to construct a variety of functionally equivalentnucleic acids, such as nucleic acids which differ in sequence but whichencode the same antibody sequence, or encode a conjugate or fusionprotein including the V_(H)H nucleic acid sequence.

Nucleic acid sequences encoding the antibodies that specifically bind aNoV polypeptide, such as a Genogroup II or Genogroup I NoV polypeptide,such as antibodies that bind a NV polypeptide, including but not limitedto VP1 or VP2 can be prepared by any suitable method including, forexample, cloning of appropriate sequences or by direct chemicalsynthesis by methods such as the phosphotriester method of Narang etal., Meth. Enzymol. 68:90-99, 1979; the phosphodiester method of Brownet al., Meth. Enzymol. 68:109-151, 1979; the diethylphosphoramiditemethod of Beaucage et al., Tetra. Lett. 22:1859-1862, 1981; the solidphase phosphoramidite triester method described by Beaucage andCaruthers, Tetra. Letts. 22(20):1859-1862, 1981, for example, using anautomated synthesizer as described in, for example, Needham-VanDevanteret al., Nucl. Acids Res. 12:6159-6168, 1984; and, the solid supportmethod of U.S. Pat. No. 4,458,066. Chemical synthesis produces a singlestranded oligonucleotide. This can be converted into double stranded DNAby hybridization with a complementary sequence or by polymerization witha DNA polymerase using the single strand as a template. One of skillwould recognize that while chemical synthesis of DNA is generallylimited to sequences of about 100 bases, longer sequences may beobtained by the ligation of shorter sequences.

Exemplary nucleic acids can be prepared by cloning techniques. Examplesof appropriate cloning and sequencing techniques, and instructionssufficient to direct persons of skill through many cloning exercises arefound in Sambrook et al., supra, Berger and Kimmel (eds.), supra, andAusubel, supra. Product information from manufacturers of biologicalreagents and experimental equipment also provide useful information.Such manufacturers include the SIGMA Chemical Company (Saint Louis,Mo.), R&D Systems (Minneapolis, Minn.), Pharmacia Amersham (Piscataway,N.J.), CLONTECH Laboratories, Inc. (Palo Alto, Calif.), Chem GenesCorp., Aldrich Chemical Company (Milwaukee, Wis.), Glen Research, Inc.,GIBCO BRL Life Technologies, Inc. (Gaithersburg, Md.), FlukaChemica-Biochemika Analytika (Fluka Chemie AG, Buchs, Switzerland),Invitrogen (Carlsbad, Calif.), and Applied Biosystems (Foster City,Calif.), as well as many other commercial sources known to one of skill.

Nucleic acids can also be prepared by amplification methods.Amplification methods include polymerase chain reaction (PCR), theligase chain reaction (LCR), the transcription-based amplificationsystem (TAS), the self-sustained sequence replication system (3SR). Awide variety of cloning methods, host cells, and in vitro amplificationmethodologies are well known to persons of skill.

Any of the nucleic acids encoding any of the antibodies, variabledomains, or CDRs disclosed herein (or fragment thereof) can be expressedin a recombinantly engineered cell such as bacteria, plant, yeast,insect and mammalian cells. These antibodies can be expressed asindividual variable domain or can be expressed as a fusion protein.Additionally, all the V_(H)H antibodies may be easily expressed inseveral biotechnological platforms such as transgenic rice or the milkof a transgenic cow.

An immunoadhesin can also be expressed. The nucleic acid sequences canoptionally encode a leader sequence.

It is expected that those of skill in the art are knowledgeable in thenumerous expression systems available for expression of proteinsincluding E. coli, other bacterial hosts, yeast, and various highereukaryotic cells such as the COS, CHO, HeLa and myeloma cell lines. Oncethe expression vector is transferred to a host cell by conventionaltechniques, the transfected cells are then cultured by conventionaltechniques, such as to produce an antibody. Thus, host cells areprovided containing a polynucleotide encoding a V_(H)H antibody fragmentthereof, or portion thereof, operably linked to a heterologous promoter.Mammalian cell lines available as hosts for expression of recombinantantibodies are well known in the art and include many immortalized celllines available from the American Type Culture Collection (ATCC),including but not limited to Chinese hamster ovary (CHO) cells, HeLacells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), humanhepatocellular carcinoma cells (e.g., Hep G2), human epithelial kidney293 cells, and a number of other cell lines. Additional cell lines whichmay be used as hosts for expression of recombinant antibodies include,but are not limited to, insect cells (e.g. Sf21/Sf9, Trichoplusia niBti-Tn5b1-4) or yeast cells (e.g. S. cerevisiae, Pichia, U.S. Pat. No.7,326,681), plant cells (US Published Patent Application No.20080066200); and chicken cells (PCT Publication No. WO2008142124).

Mammalian cell lines available as hosts for expression of recombinantantibodies are well known in the art and include many immortalized celllines available from the American Type Culture Collection (ATCC),including but not limited to Chinese hamster ovary (CHO) cells, HeLacells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), humanhepatocellular carcinoma cells (e.g., Hep G2), human epithelial kidney293 cells, and a number of other cell lines. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins and gene products. Appropriatecell lines or host systems can be chosen to ensure the correctmodification and processing of the antibody or portion thereofexpressed. To this end, eukaryotic host cells which possess the cellularmachinery for proper processing of the primary transcript,glycosylation, and phosphorylation of the gene product may be used. Suchmammalian host cells include but are not limited to CHO, VERY, BHK,Hela, COS, MDCK, 293, 3T3, W138, BT483, Hs578T, HTB2, BT2O and T47D, NS0(a murine myeloma cell line that does not endogenously produce anyfunctional immunoglobulin chains), SP20, CRL7O3O and HsS78Bst cells. Inone embodiment, human cell lines are of use. In one embodiment, thehuman cell line PER.C6. (Crucell, Netherlands) can be used. Additionalcell lines which may be used as hosts for expression of recombinantantibodies include, but are not limited to, insect cells (e.g. Sf21/Sf9,Trichoplusia ni Bti-Tn5b1-4) or yeast cells (e.g. S. cerevisiae, Pichia,U.S. Pat. No. 7,326,681), plant cells (US Published Patent ApplicationNo. 20080066200); and chicken cells (PCT Publication No. WO2008142124).

The host cell can be a gram positive bacteria including, but not limitedto, Bacillus, Streptococcus, Streptomyces, Staphylococcus, Enterococcus,Lactobacillus, Lactococcus, Clostridium, Geobacillus, andOceanobacillus. Methods for expressing protein in gram positivebacteria, such as Lactobaccillus are well known in the art, see forexample, U.S. Published Patent Application No. 20100/080774. Expressionvectors for lactobacillus are described, for example in U.S. Pat. Nos.6,100,388, and 5,728,571. Leader sequences can be included forexpression in Lactobacillus. Gram negative bacteria include, but notlimited to, E. coli, Pseudomonas, Salmonella, Campylobacter,Helicobacter, Flavobacterium, Fusobacterium, Ilyobacter, Neisseria, andUreaplasma.

One or more DNA sequences encoding the antibody or fragment thereof canbe expressed in vitro by DNA transfer into a suitable host cell. Theterm also includes any progeny of the subject host cell. It isunderstood that all progeny may not be identical to the parental cellsince there may be mutations that occur during replication. Methods ofstable transfer, meaning that the foreign DNA is continuously maintainedin the host, are known in the art.

The expression of nucleic acids encoding the isolated proteins describedherein can be achieved by operably linking the DNA to a promoter (whichis either constitutive or inducible), followed by incorporation into anexpression cassette. The promoter can be any promoter of interest,including a cytomegalovirus promoter and a human T cell lymphotrophicvirus promoter (HTLV)-1. Optionally, an enhancer, such as acytomegalovirus enhancer, is included in the construct. The cassettescan be suitable for replication and integration in either prokaryotes oreukaryotes. Typical expression cassettes contain specific sequencesuseful for regulation of the expression of the DNA encoding the protein.For example, the expression cassettes can include appropriate promoters,enhancers, transcription and translation terminators, initiationsequences, a start codon (i.e., ATG) in front of a protein-encodinggene, splicing signal for introns, sequences for the maintenance of thecorrect reading frame of that gene to permit proper translation of mRNA,and stop codons. The vector can encode a selectable marker, such as amarker encoding drug resistance (for example, ampicillin or tetracyclineresistance).

To obtain high level expression of a cloned gene, it is desirable toconstruct expression cassettes which contain, at the minimum, a strongpromoter to direct transcription, a ribosome binding site fortranslational initiation (internal ribosomal binding sequences), and atranscription/translation terminator. For E. coli, this includes apromoter such as the T7, trp, lac, or lambda promoters, a ribosomebinding site, and preferably a transcription termination signal. Foreukaryotic cells, the control sequences can include a promoter and/or anenhancer derived from, for example, an immunoglobulin gene, HTLV, SV40or cytomegalovirus, and a polyadenylation sequence, and can furtherinclude splice donor and/or acceptor sequences (for example, CMV and/orHTLV splice acceptor and donor sequences). The cassettes can betransferred into the chosen host cell by well-known methods such astransformation or electroporation for E. coli and calcium phosphatetreatment, electroporation or lipofection for mammalian cells. Cellstransformed by the cassettes can be selected by resistance toantibiotics conferred by genes contained in the cassettes, such as theamp, gpt, neo and hyg genes.

When the host is a eukaryote, such methods of transfection of DNA ascalcium phosphate coprecipitates, conventional mechanical proceduressuch as microinjection, electroporation, insertion of a plasmid encasedin liposomes, or virus vectors may be used. Eukaryotic cells can also becotransformed with polynucleotide sequences encoding the antibody,labeled antibody, or functional fragment thereof, and a second foreignDNA molecule encoding a selectable phenotype, such as the herpes simplexthymidine kinase gene. Another method is to use a eukaryotic viralvector, such as simian virus 40 (SV40) or bovine papilloma virus, totransiently infect or transform eukaryotic cells and express the protein(see for example, Eukaryotic Viral Vectors, Cold Spring HarborLaboratory, Gluzman ed., 1982). One of skill in the art can readily usean expression systems such as plasmids and vectors of use in producingproteins in cells including higher eukaryotic cells such as the COS,CHO, HeLa and myeloma cell lines.

The disclosed antibodies and antibody fragments can be produced in amonocot plant, such as rice. In general, expression vectors for useinclude operably linked components that constitute a chimeric gene: apromoter from the gene of a maturation-specific monocot plant storageprotein, a first DNA sequence, operably linked to the promoter, encodinga monocot plant seed-specific signal sequence (such as an N-terminalleader sequence or a C-terminal trailer sequence) capable of targeting apolypeptide linked thereto to an endosperm cell, such as to anendosperm-cell organelle, for example to a protein storage body, and asecond DNA sequence, linked in translation frame with the first DNAsequence, encoding the V_(H)H antibody or antigen binding fragmentthereof. The signal sequence can be cleaved from the V_(H)H antibody orantigen binding fragment thereof in the plant cell. The chimeric gene,in turn, is typically placed in a suitable plant-transformation vectorhaving (i) companion sequences upstream and/or downstream of thechimeric gene which are of plasmid or viral origin and provide necessarycharacteristics to the vector to permit the vector to move DNA frombacteria to the desired plant host; (ii) a selectable marker sequence;and (iii) a transcriptional termination region generally at the oppositeend of the vector from the transcription initiation regulatory region.

The chimeric gene, in turn, is typically placed in a suitableplant-transformation vector having (i) companion sequences upstreamand/or downstream of the chimeric gene which are of plasmid or viralorigin and provide necessary characteristics to the vector to permit thevector to move DNA from bacteria to the desired plant host; (ii) aselectable marker sequence; and (iii) a transcriptional terminationregion generally at the opposite end of the vector from thetranscription initiation regulatory region.

Numerous types of appropriate expression vectors, and suitableregulatory sequences are known in the art for a variety of plant hostcells. The promoter region is chosen to be regulated in a mannerallowing for induction under seed-maturation conditions. In one aspectof this embodiment of the invention, the expression construct includes apromoter which exhibits specifically upregulated activity during seedmaturation. Promoters for use in the invention are typically derivedfrom cereals such as rice, barley, wheat, oat, rye, corn, millet,triticale or sorghum. Examples of such promoters include thematuration-specific promoter region associated with one of the followingmaturation-specific monocot plant storage proteins: rice glutelins,oryzins, and prolamines, barley hordeins, wheat gliadins and glutelins,maize zeins and glutelins, oat glutelins, and sorghum kafirins, milletpennisetins, and rye secalins. Other promoters suitable for expressionin maturing seeds include the barley endosperm-specific B1-hordeinpromoter, GluB-2 promoter, Bx7 promoter, Gt3 promoter, GluB-1 promoterand Rp-6 promoter, particularly if these promoters are used inconjunction with transcription factors. In some embodiments, theexpression of the nucleic acid encoding the V_(H)H antibody or antigenbinding fragment thereof from a promoter that is preferentiallyexpressed in plant seed tissue. Examples of such promoter sequencesinclude those sequences derived from sequences encoding plant storageprotein genes or from genes involved in fatty acid biosynthesis inoilseeds. Non-limiting examples are a glutelin (Gt1) promoter, whichprovides gene expression in the outer layer of the endosperm, and aglobulin (Gib) promoter, which provides gene expression in the center ofthe endosperm. Promoter sequences for regulating transcription ofoperably linked gene coding sequences include naturally-occurringpromoters, or regions thereof capable of directing seed-specifictranscription, and hybrid promoters, which combine elements of more thanone promoter. In some examples, the promoter is native to the same plantspecies as the plant cells into which the chimeric nucleic acidconstruct is to be introduced. In other embodiments, the promoter isheterologous to the plant host cell. In other embodiments, aseed-specific promoter from one type of monocot may be used regulatetranscription of a nucleic acid coding sequence from a different monocotor a non-cereal monocot. See, for example, U.S. Published PatentApplication No. 2012/0195883 and U.S. Published Patent Application No.2008/0318277.

In some embodiments the monocot plant can be stably transformed with achimeric gene having (i) a seed maturation-specific promoter, (ii)operably linked to said promoter, a leader DNA sequence encoding amonocot seed-specific transit sequence capable of targeting a linkedpolypeptide to an endosperm-cell organelle, and (iii) a protein-codingsequence encoding a V_(H)H or antigen binding fragment thereof, (b)cultivating the transformed plant under seed-maturation conditions, (c)harvesting the seeds from the cultivated plant, (d) extracting theharvested seeds with an aqueous solution, thereby obtaining an extractof water soluble plant components comprising at least 3% by totalprotein weight of the V_(H)H or antigen binding fragment thereof, (e)purifying the V_(H)H or antigen binding fragment thereof from theaqueous solution. Plant cells or tissues are transformed with expressionconstructs (heterologous nucleic acid constructs, e.g., plasmid DNA intowhich the gene of interest has been inserted) using a variety ofstandard techniques, including, but not limited to, the use ofAgrobacterium.

Suitable vectors and methods for expressing the V_(H)H antibody orantigen binding fragment thereof in transgenic plants are disclosed, forexample, in U.S. Published Patent Application No. 2012/0195883 and U.S.Published Patent Application No. 2008/0318277, which are incorporatedherein by reference.

Modifications can be made to a nucleic acid encoding a polypeptidedescribed herein without diminishing its biological activity. Somemodifications can be made to facilitate the cloning, expression, orincorporation of the targeting molecule into a fusion protein. Suchmodifications are well known to those of skill in the art and include,for example, termination codons, a methionine added at the aminoterminus to provide an initiation, site, additional amino acids placedon either terminus to create conveniently located restriction sites,codon optimization, or additional amino acids (such as poly His) to aidin purification steps. In addition to recombinant methods, theimmunoconjugates, effector moieties, and antibodies of the presentdisclosure can also be constructed in whole or in part using standardpeptide synthesis well known in the art.

Once expressed, the recombinant immunoconjugates, antibodies, and/oreffector molecules can be purified according to standard procedures ofthe art of the expression system used, including ammonium sulfateprecipitation, affinity columns, column chromatography, preparative gelfiltration, and the like (see, generally, R. Scopes, PROTEINPURIFICATION, Springer-Verlag, N.Y., 1982). The antibodies,immunoconjugates and effector molecules need not be 100% pure. In someembodiments, the V_(H)H antibodies or antigen binding fragment thereofcan be purified from seed extracts (see, generally, Greenham andAltosaar, Methods in Mol. Biol. 956: 311, 2013; Broz et al., Gen. Eng.Biotechnol. News 33(4), 2013; Nandi et al., Trans. Res. 14: 237, 2005).The antibody can be purified from a seed product by methods that includegrinding, filtration, heat, pressure, salt extraction, evaporation, orchromatography (see U.S. Published Patent Application No. 20120195883).Once purified, partially or to homogeneity as desired, if to be usedtherapeutically, the polypeptides should be substantially free ofendotoxin.

Methods for expression of antibodies and/or refolding to an appropriateactive form, including single domain or single chain antibodies, frombacteria such as E. coli, have been described and are well-known and areapplicable to the antibodies disclosed herein. See, Buchner et al.,Anal. Biochem. 205:263-270, 1992; Pluckthun, Biotechnology 9:545, 1991;Huse et al., Science 246:1275, 1989 and Ward et al., Nature 341:544,1989.

In addition to recombinant methods, the antibodies, labeled antibodiesand functional fragments thereof that are disclosed herein can also beconstructed in whole or in part using standard peptide synthesis. Solidphase synthesis of the polypeptides of less than about 50 amino acids inlength can be accomplished by attaching the C-terminal amino acid of thesequence to an insoluble support followed by sequential addition of theremaining amino acids in the sequence. Techniques for solid phasesynthesis are described by Barany & Merrifield, The Peptides: Analysis,Synthesis, Biology. Vol. 2: Special Methods in Peptide Synthesis, PartA. pp. 3-284; Merrifield et al., J. Am. Chem. Soc. 85:2149-2156, 1963,and Stewart et al., Solid Phase Peptide Synthesis, 2nd ed., Pierce Chem.Co., Rockford, Ill., 1984. Proteins of greater length may be synthesizedby condensation of the amino and carboxyl termini of shorter fragments.Methods of forming peptide bonds by activation of a carboxyl terminalend (such as by the use of the coupling reagent N,N′-dicylohexylcarbodimide) are well known in the art. Once an antibodymolecule has been produced, it may be purified by any method known inthe art for purification of an immunoglobulin molecule, for example, bychromatography (e.g., ion exchange, affinity, and sizing columnchromatography), centrifugation, differential solubility, or by anyother standard technique for the purification of proteins. Further, theantibodies or fragments thereof may be fused to heterologous polypeptidesequences (referred to herein as “tags”) described above or otherwiseknown in the art to facilitate purification.

Compositions and Therapeutic Methods

Methods are disclosed herein for the prevention or treatment of a NoVinfection. Included within the NoVs are at least 5 genogroups (GI-GV),separated by nucleic acid and amino acid sequences, which comprise 15genetic clusters. Methods are provided for the prevention and/ortreatment of NoVs from any of these groups. Examples of NoVs include,but are not limited to, NV (NV, see, for example, GENBANK® Accession No.M87661, NP_056821), P7-587 virus (see, for example, GENBANK® AccessionNo. FJ384783), Southampton virus (SHV, see, for example, GENBANK®Accession No. L07418), Desert Shield virus (DSV, see, for example,GENBANK® Accession No. U04469), Hesse virus (HSV, see, for example,GENBANK® Accession No. AF093797), Chiba virus (CHV, see, for example,GENBANK® Accession No. AB042808), Hawaii virus (HV, see, for example,GENBANK® Accession No. U07611), Snow Mountain virus (SMV, see, forexample, GENBANK® Accession No. U70059), Toronto virus (TV, see forexample, GENBANK® Accession No. U02030), Bristol virus (BV, see forexample, GENBANK® Accession No. X76716), Jena virus (JV, see, forexample, GENBANK® Accession No. AJ011099), MD145-12 virus (MD145-12,see, for example, GENBANK® Accession No. AY032605), Aichi virus(AV124-89, see, for example, GENBANK® Accession No. AB031013),Camberwell (CV, see, for example, GENBANK® Accession No. AF145896),Lordsdale virus (LV, see, for example, GENBANK® Accession No. X86557),Grimsby virus (GrV, see, for example, GENBANK® Accession No. AJ004864),Mexico virus (MXV, see, for example, GENBANK® Accession No. U22498),Boxer (see, for example, GENBANK® Accession No. AF538679), C59 (see, forexample, GENBANK® Accession No. AF435807), VA98115 (see, for example,GENBANK® Accession No. AY038598), BUDS (see, for example, GENBANK®Accession No. AY660568), MOH (see, for example, GENBANK® Accession No.AF397156), Parris Island (PiV; see, for example, GENBANK® Accession No.AY652979), VA98387 (see, for example, GENBANK® Accession No. AY038600),VA97207 (see, for example, GENBANK® Accession No. AY038599), andOperation Iraqi Freedom (see, for example, OIF, GENBANK® Accession No.AY675554). In some embodiments, methods are provided for the treatmentand/or prevention of Genogroup I NV. In some embodiments, methods areprovided for the treatment and/or prevention of Genogroup II NV. In somenon-limiting examples, methods are provided for the treatment and/orprevention of a NV infection.

Prevention can include inhibition of infection with the NoV, such as aNorwalk virus or MD2004 virus. In some embodiments, the methods includecontacting a cell with an effective amount of one or more of theantibodies disclosed herein. In some embodiments, the antibodyspecifically binds VP1, or an antigen binding fragment thereof. Themethod can also include administering to a subject a therapeuticallyeffective amount of a V_(H)H monoclonal antibody, or a nucleic acidencoding the antibody. Neutralizing MAbs against NoVs can be used asemergency prophylaxis to protect individuals in the proximity of adeveloping NoV outbreak, or when encountering an increased risk ofexposure. Thus, the methods can include selecting a subject at risk ofexposure to a NoV. The antibodies can also be used to clear a chronicinfection and reduce viral load in a severe case of NoV gastroenteritis.

In other embodiments, methods are disclosed for ameliorating one or moresymptoms associated with a NoV infection, such as a Genogroup I orGenogroup II NoV infection, for example, a Norwalk virus infection.Generally, the method includes administering an antibody or antibody(antigen-binding) fragment that specifically binds a NoV polypeptide,such as a Norwalk virus polypeptide. The antibody can specifically bindVP1. In some embodiments, the disclosed antibodies can be used intreatment to alleviate chronic NoV gastroenteritis in debilitated orimmunocompromised individuals. Thus, the method can include selecting asubject with gastroenteritis. In some examples, the subject isimmunocompromised. In some embodiments, the subject is a prematureinfant. In some embodiments, an individual is debilitated bychemotherapy for cancer. In some embodiments, an elderly individual withprolonged NoV disease can be treated.

The NoV infection does not need to be completely eliminated for thecomposition to be effective. For example, a composition can decrease theNoV infection in a population by a desired amount, for example by atleast 10%, at least 20%, at least 50%, at least 60%, at least 70%, atleast 80%, at least 90%, at least 95%, at least 98%, or 100%, ascompared to the rate of infection in the absence of the composition. Inaddition, a composition can decrease viral titer by at least 10%, atleast 20%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 95%, at least 98%, or 100% in a subject.

In example, the subject is also administered an effective amount of anadditional agent, such as anti-viral agent. The methods can includeadministration of one on more additional agents known in the art. Thesubject can hydrated and administered balancing electrolytes.

A therapeutically effective amount of a NoV-specific, such as aGenogroup I or Genogroup II-specific (or Norwalk virus specific) V_(H)Hmonoclonal antibody or antigen binding fragment (or the nucleic acidencoding the antibody or antigen binding fragment), or nucleic acid,will depend upon the severity of the disease and/or infection and thegeneral state of the patient's health. A therapeutically effectiveamount of the antibody can provide either subjective relief of asymptom(s) or an objectively identifiable improvement as noted by theclinician or other qualified observer. As noted above, thesecompositions can be administered in conjunction with another therapeuticagent, either simultaneously or sequentially. For any application, theantibody, antigen binding fragment, or nucleic acid encoding theantibody or antigen binding fragment can be combined with anti-viraltherapy.

In one embodiment, administration of the monoclonal antibody (or nucleicacid encoding the antibody), such as the V_(H)H monoclonal antibody,results in a reduction in the establishment of a virus infection and/orreducing subsequent disease progression in a subject. A reduction in theestablishment of NoV infection, such as a Norwalk virus infection, or aMD2004 virus infection, and/or a reduction in subsequent diseaseprogression can encompass a statistically significant reduction in viralactivity. In some embodiments, the method reduces disease in a subject.In some embodiments, methods are disclosed for treating a subject with aNoV infection, such as a Norwalk virus infection or a MD2004 virusinfection. These methods include administering to the subject atherapeutically effective amount of an antibody, or a nucleic acidencoding the antibody, thereby preventing or treating the viralinfection.

Single or multiple administrations of the compositions including theantibody, antigen binding fragment, or nucleic acid encoding theantibody or antigen binding fragment, that are disclosed herein, areadministered depending on the dosage and frequency as required andtolerated by the patient. In any event, the composition should provide asufficient quantity of at least one of the antibodies disclosed hereinto effectively treat the patient. The dosage can be administered oncebut may be applied periodically until either a therapeutic result isachieved or until side effects warrant discontinuation of therapy. Inone example, a dose of the antibody is infused for thirty minutes everyother day. In this example, about one to about ten doses can beadministered, such as three or six doses can be administered every otherday. In a further example, a continuous infusion is administered forabout five to about ten days. The subject can be treated at regularintervals, such as monthly, until a desired therapeutic result isachieved. Generally, the dose is sufficient to treat or amelioratesymptoms or signs of disease without producing unacceptable toxicity tothe patient.

Compositions are provided that include one or more of the V_(H)Hmonoclonal antibodies that specifically bind a NoV polypeptide, such asa Genogroup I or Genogroup II NoV polypeptide, such as a Norwalk viruspolypeptide (for example an antibody that specifically binds VP1), orantigen binding fragments of any of these antibodies, and nucleic acidsencoding these antibodies (and antigen binding fragments) that aredisclosed herein in a carrier. The compositions can be prepared in unitdosage forms for administration to a subject. The amount and timing ofadministration are at the discretion of the treating physician toachieve the desired purposes. The antibody and/or nucleic acid can beformulated for systemic or local administration. In one example, theantibody and/or nucleic acid is formulated for parenteraladministration, such as intravenous administration. In some embodiments,administration is intramuscular.

Compositions also can be formulated for enteric delivery. Variousstudies have been made on a method of releasing or delivering a drugselectively to a specific site in the intestine. In addition to classicmethods of using enteric-coated preparations or sustained releasepreparations (Chemical & Pharmaceutical Bulletin, 40, 3035-3041, 1992),enteric-coated sustained-release preparations and time-limited releaseenteric-coated preparations have been proposed (Japanese Patent No.3185206, and PCT Publication No. WO 01/23000).

Active ingredients can also be entrapped in microcapsules prepared, forexample, by coacervation techniques or by interfacial polymerization,for example, hydroxymethylcellulose or gelatin-microcapsule andpoly-(methylmethacylate) microcapsule, respectively, in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules) or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences16th edition, Osol, A. Ed. (1980). Specifically, liposomes containingthe immunogens or antibodies can be prepared by such methods asdescribed in Epstein et al., Proc. Natl. Acad. Sci. USA, 82:3688, 1985;Hwang et al., Proc. Natl. Acad. Sci. USA, 77:4030, 1980; and U.S. Pat.Nos. 4,485,045 and 4,544,545. Liposomes with enhanced circulation timeare disclosed in U.S. Pat. No. 5,013,556. The everse-phase evaporationmethod can be used with a lipid composition comprisingphosphatidylcholine, cholesterol and PEG-derivatizedphosphatidylethanolamine (PEG-PE). Liposomes are extruded throughfilters of defined pore size to yield liposomes with the desireddiameter. The antibodies disclosed herein can be conjugated to theliposomes as described, for example, in Martin et al., J. Biol. Chem.,257:286-288, 1982, via a disulfide interchange reaction.

The compositions for administration can include a solution of theantibody, such as the V_(H)H monoclonal antibody or antigen bindingfragment dissolved in a pharmaceutically acceptable carrier, such as anaqueous carrier. A variety of aqueous carriers can be used, for example,buffered saline and the like. These compositions may be sterilized byconventional, well known sterilization techniques. The compositions maycontain pharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions such as pH adjusting and bufferingagents, toxicity adjusting agents and the like, for example, sodiumacetate, sodium chloride, potassium chloride, calcium chloride, sodiumlactate and the like. The concentration of antibody in theseformulations can vary widely, and will be selected primarily based onfluid volumes, viscosities, body weight and the like in accordance withthe particular mode of administration selected and the subject's needs.In some embodiments, administration is intravenous.

Controlled-release parenteral formulations can be made as implants, oilyinjections, or as particulate systems. For a broad overview of proteindelivery systems see, Banga, Therapeutic Peptides and Proteins:Formulation, Processing, and Delivery Systems, Technomic PublishingCompany, Inc., Lancaster, Pa., 1995. Particulate systems includemicrospheres, microparticles, microcapsules, nanocapsules, nanospheres,and nanoparticles. Microcapsules contain the therapeutic protein, suchas a cytotoxin or a drug, as a central core. In microspheres thetherapeutic is dispersed throughout the particle. Particles,microspheres, and microcapsules smaller than about 1 μm are generallyreferred to as nanoparticles, nanospheres, and nanocapsules,respectively. Capillaries have a diameter of approximately 5 μm so thatonly nanoparticles are administered intravenously. Microparticles aretypically around 100 μm in diameter and are administered subcutaneouslyor intramuscularly. See, for example, Kreuter, Colloidal Drug DeliverySystems, J. Kreuter, ed., Marcel Dekker, Inc., New York, N.Y., pp.219-342, 1994; and Tice & Tabibi, Treatise on Controlled Drug Delivery,A. Kydonieus, ed., Marcel Dekker, Inc. New York, N.Y., pp. 315-339,1992.

Polymers can be used for ion-controlled release of the antibodycompositions disclosed herein. Various degradable and nondegradablepolymeric matrices for use in controlled drug delivery are known in theart (Langer, Accounts Chem. Res. 26:537-542, 1993). For example, theblock copolymer, polaxamer 407, exists as a viscous yet mobile liquid atlow temperatures but forms a semisolid gel at body temperature. It hasbeen shown to be an effective vehicle for formulation and sustaineddelivery of recombinant interleukin-2 and urease (Johnston et al.,Pharm. Res. 9:425-434, 1992; and Pec et al., J. Parent. Sci. Tech.44(2):58-65, 1990). Alternatively, hydroxyapatite has been used as amicrocarrier for controlled release of proteins (Ijntema et al., Int. J.Pharm. 112:215-224, 1994). In yet another aspect, liposomes are used forcontrolled release as well as drug targeting of the lipid-capsulateddrug (Betageri et al., Liposome Drug Delivery Systems, TechnomicPublishing Co., Inc., Lancaster, Pa., 1993). Numerous additional systemsfor controlled delivery of therapeutic proteins are known (see U.S. Pat.Nos. 5,055,303; 5,188,837; 4,235,871; 4,501,728; 4,837,028; 4,957,735;5,019,369; 5,055,303; 5,514,670; 5,413,797; 5,268,164; 5,004,697;4,902,505; 5,506,206; 5,271,961; 5,254,342 and 5,534,496).

A typical pharmaceutical composition for intravenous administrationincludes about 0.1 to 10 mg/kg of antibody per day, or 0.5 to 15 mg/kgof antibody per day. Dosages from 0.1 up to about 100 mg/kg per subjectper day may be used, particularly if the agent is administered to asecluded site and not into the circulatory or lymph system, such as intoa body cavity or into a lumen of an organ. Exemplary doses include 1 to10 mg/kg, such as 2 to 8 mg/kg, such as 3 to 6 mg/kg. Actual methods forpreparing administrable compositions will be known or apparent to thoseskilled in the art and are described in more detail in such publicationsas Remington's Pharmaceutical Science, 19th ed., Mack PublishingCompany, Easton, Pa. (1995). Administration can be oral.

Antibodies may be provided in lyophilized form and rehydrated withsterile water before administration, although they are also provided insterile solutions of known concentration. The antibody solution is thenadded to an infusion bag containing 0.9% sodium chloride, USP, andtypically administered at a dosage of from 0.1 to 10 mg/kg or 0.5 to 15mg/kg of body weight. Exemplary doses include 1 to 10 mg/kg, such as 2to 8 mg/kg, such as 3 to 6 mg/kg. Considerable experience is availablein the art in the administration of antibody drugs, which have beenmarketed in the U.S. since the approval of RITUXAN® in 1997. Antibodiescan be administered by slow infusion, rather than in an intravenous pushor bolus. In one example, a higher loading dose is administered, withsubsequent, maintenance doses being administered at a lower level. Forexample, an initial loading dose of 4 mg/kg may be infused over a periodof some 90 minutes, followed by weekly maintenance doses for 4-8 weeksof 2 mg/kg infused over a 30 minute period if the previous dose was welltolerated.

A therapeutically effective amount of a nucleic acid encoding theantibody or an antigen binding fragment thereof can be administered to asubject in need thereof. One approach to administration of nucleic acidsis direct immunization with plasmid DNA, such as with a mammalianexpression plasmid. The nucleotide sequence encoding the antibody orfragment thereof can be placed under the control of a promoter toincrease expression of the molecule. Immunization by nucleic acidconstructs is well known in the art and taught, for example, in U.S.Pat. Nos. 5,643,578, and 5,593,972 and 5,817,637. U.S. Pat. No.5,880,103 describes several methods of delivery of nucleic acids to anorganism. The methods include liposomal delivery of the nucleic acids.

In another approach to using nucleic acids, an antibody or antigenbinding fragment thereof can also be expressed by attenuated viral hostsor vectors or bacterial vectors, which can be administered to a subject.Recombinant vaccinia virus, adeno-associated virus (AAV), herpes virus,retrovirus, cytomegalovirus, poxvirus or other viral vectors can be usedto express the antibody. For example, vaccinia vectors are described inU.S. Pat. No. 4,722,848. BCG (Bacillus Calmette Guerin) provides anothervector for expression of the disclosed antibodies (see Stover, Nature351:456-460, 1991).

In one embodiment, a nucleic acid encoding the antibody or an antigenbinding fragment thereof is introduced directly into cells. For example,the nucleic acid can be loaded onto gold microspheres by standardmethods and introduced into the skin by a device such as Bio-Rad'sHeliosä Gene Gun. The nucleic acids can be “naked,” consisting ofplasmids under control of a strong promoter.

Typically, the DNA is injected into muscle, although it can also beinjected directly into other sites. Dosages for injection are usuallyaround 0.5 mg/kg to about 50 mg/kg, and typically are about 0.005 mg/kgto about 5 mg/kg (see, e.g., U.S. Pat. No. 5,589,466).

In some examples, a subject is administered the DNA encoding theantibody or antibody binding fragments thereof to provide in vivoantibody production, for example using the cellular machinery of thesubject Immunization by nucleic acid constructs is well known in the artand taught, for example, in U.S. Pat. Nos. 5,643,578, and 5,593,972 and5,817,637. U.S. Pat. No. 5,880,103 describes several methods of deliveryof nucleic acids encoding to an organism. The methods include liposomaldelivery of the nucleic acids. Such methods can be applied to theproduction of an antibody, or antibody binding fragments thereof, by oneof ordinary skill in the art.

One approach to administration of nucleic acids is direct administrationwith plasmid DNA, such as with a mammalian expression plasmid. Thenucleotide sequence encoding the disclosed antibody, or antibody bindingfragments thereof, can be placed under the control of a promoter toincrease expression.

In another approach to using nucleic acids, a disclosed antibody, orantibody binding fragments thereof can also be expressed by attenuatedviral hosts or vectors or bacterial vectors. Recombinant vaccinia virus,adeno-associated virus (AAV), herpes virus, retrovirus, cytomegalovirusor other viral vectors can be used to express the antibody. For example,vaccinia vectors and methods useful protocols are described in U.S. Pat.No. 4,722,848. BCG (Bacillus Calmette Guerin) provides another vectorfor expression of the disclosed antibodies (see Stover, Nature351:456-460, 1991).

In one embodiment, a nucleic acid encoding a disclosed antibody, orantibody binding fragments thereof, is introduced directly into cells.For example, the nucleic acid can be loaded onto gold microspheres bystandard methods and introduced into the skin by a device such asBio-Rad's HELIOS™ Gene Gun. The nucleic acids can be “naked,” consistingof plasmids under control of a strong promoter.

Typically, the DNA is injected into muscle, although it can also beinjected directly into other sites. Dosages for injection are usuallyaround 0.5 μg/kg to about 50 mg/kg, and typically are about 0.005 mg/kgto about 5 mg/kg (see, e.g., U.S. Pat. No. 5,589,466).

Diagnostic Methods and Kits

A method is provided herein for the detection of a NoV, such as aGenogroup I or Genogroup II NoV in vitro or in vivo. The methods can beused to detect, for example, a Norwalk virus infection or a MD2004 virusinfection. In one example, the NoV is detected in a biological sample,and can be used to an infection with the virus. The method can detectthe presence of a NoV polypeptide, such as VP1. The sample can be anysample, including, but not limited to, tissue from biopsies, autopsiesand pathology specimens. Biological samples also include sections oftissues, for example, frozen sections taken for histological purposes.Biological samples further include body fluids, such as blood, serum,plasma, sputum, spinal fluid, nasopharyngeal secretions, or urine.Biological samples can include stool.

In one embodiment, methods are provided for detecting the presence of aNoV, such as a Genogroup I or Genogroup II NoV. The presence of the NoVis detected in in a sample suspected of containing the virus, whereinthe method includes contacting the sample with a V_(H)H antibodydisclosed herein, or antigen binding fragment thereof, and determiningbinding of the antibody or antigen binding fragment to the virus in thesample. Binding of the antibody to virus in the sample is indicative ofthe presence of the virus in the sample. In one embodiment, the sampleis a biological sample. In some examples, the sample is a stool sample.In other embodiments, the sample is an environmental sample. In someembodiments, the method distinguishes a particular NoV from other NoV,distinguishes a Genogroup I NoV, or distinguishes a Genogroup II NoV.

As discussed above, NoVs are divided into five distinct genogroups basedon VP1 sequence similarity. Virus strains from Genogroups I and II areresponsible for most human infections, and these genogroups are furthersubdivided into more than 25 different genotypes (Zheng et al., Virology346:312-23, 2006; Kroneman et al., Arch Virol 158(10): 2059-68, 2013.).In some embodiments, methods are provided for detecting ordistinguishing a Genogroup I and/or Genogroup II NoV. The methodincludes contacting the sample with a V_(H)H monoclonal antibodydisclosed herein, or an antigen binding fragment thereof, anddetermining binding of the antibody to the virus in the sample. In someembodiments, binding of the antibody to virus in the sample isindicative of the presence of a Genogroup NoV, such as GI.1 NV in thesample. In some examples, the sample is a stool sample. In otherembodiments, the sample is an environmental sample.

In several embodiments, a method is provided for detecting a NoVinfection, such as Norwalk virus infection in a subject. The disclosureprovides a method for detecting a NoV in a biological sample, whereinthe method includes contacting a biological sample with the antibodyunder conditions conducive to the formation of an immune complex, anddetecting the immune complex, to detect the presence of a NoVpolypeptide, such as, but not limited to, a Norwalk virus (NV)polypeptide in the biological sample. In some embodiments, NV VP1, isdetected in the biological sample. In another example, detection of thevirus in the sample confirms a diagnosis of a NoV infection, in asubject.

In some specific non-limiting examples, the V_(H)H monoclonal antibody,or fragment thereof, specifically binds a NV polypeptide, such as VP1.In these examples, the method detects a GI NV infection in the subject,such as, but not limited to, a GI.1 NV infection.

The detection of a NoV can be achieved, for example, by contacting asample to be tested, optionally along with a control sample, with theantibody under conditions that allow for formation of a complex betweenthe antibody and the virus. Complex formation is then detected (e.g.,using an ELISA). When using a control sample along with the test sample,a complex is detected in both samples and any statistically significantdifference in the formation of complexes between the samples isindicative of the presence of a NoV in the test sample.

In some embodiments, the disclosed antibodies are used to test vaccines.For example to test if a vaccine composition can induce neutralizingantibodies to one or both Genogroups of NoV. Thus provided herein is amethod for detecting testing a vaccine, wherein the method includescontacting a sample containing the vaccine, such as an NoV polypeptide,for example a Norwalk virus polypeptide, with the antibody underconditions conducive to the formation of an immune complex, anddetecting the immune complex, to confirm the vaccine will be effective.In one example, the detection of the immune complex in the sampleindicates that vaccine component, such as such as a NoV antigen (forexample a Norwalk virus antigen, such as VP1) assumes a conformationcapable of inducing neutralizing antibodies.

In some embodiments of the disclosed methods, an antibody is directlylabeled with a detectable label. In another embodiment, the antibodythat binds the NoV (the first antibody) is unlabeled and a secondantibody or other molecule that can bind the antibody that binds the NoVpolypeptide is utilized. As is well known to one of skill in the art, asecond antibody is chosen that is able to specifically bind the specificspecies and class of the first antibody. For example, if the firstantibody is a llama V_(H)H, then the secondary antibody may be ananti-V_(H)H made in another species such as guinea pig or rabbit.

Suitable labels for the antibody or secondary antibody are describedabove, and include various enzymes, prosthetic groups, fluorescentmaterials, luminescent materials, magnetic agents and radioactivematerials. Non-limiting examples of suitable enzymes include horseradishperoxidase, alkaline phosphatase, beta-galactosidase, oracetylcholinesterase. Non-limiting examples of suitable prosthetic groupcomplexes include streptavidin/biotin and avidin/biotin. Non-limitingexamples of suitable fluorescent materials include umbelliferone,fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin. Anon-limiting exemplary luminescent material is luminol; a non-limitingexemplary a magnetic agent is gadolinium, and non-limiting exemplaryradioactive labels include ¹²⁵I, ¹³¹I, ³⁵S or ³H.

The immunoassays and method disclosed herein can be used for a number ofpurposes. Kits for detecting n NoV polypeptide will typically comprise aV_(H)H antibody that binds a NoV polypeptide, such as a Norwalk viruspolypeptide, for example, any of the antibodies disclosed herein. Insome embodiments, a V_(H)H antibody fragment is included in the kit. Ina further embodiment, the V_(H)H antibody is labeled (for example, witha fluorescent, radioactive, or an enzymatic label).

In one embodiment, a kit includes instructional materials disclosingmeans of use. The instructional materials may be written, in anelectronic form (such as a computer diskette or compact disk) or may bevisual (such as video files). The kits may also include additionalcomponents to facilitate the particular application for which the kit isdesigned. Thus, for example, the kit may additionally contain means ofdetecting a label (such as enzyme substrates for enzymatic labels,filter sets to detect fluorescent labels, appropriate secondary labelssuch as a secondary antibody, or the like). The kits may additionallyinclude buffers and other reagents routinely used for the practice of aparticular method. Such kits and appropriate contents are well known tothose of skill in the art.

In one embodiment, the diagnostic kit comprises an immunoassay. Althoughthe details of the immunoassays may vary with the particular formatemployed, the method of detecting the NoV polypeptide, such as a Norwalkvirus polypeptide, such as VP1 and/or VP2 in a biological samplegenerally includes the steps of contacting the biological sample with anantibody which specifically reacts, under immunologically reactiveconditions, to the viral polypeptide. The antibody is allowed tospecifically bind under immunologically reactive conditions to form animmune complex, and the presence of the immune complex (bound antibody)is detected directly or indirectly.

The following examples are provided to illustrate certain particularfeatures and/or embodiments. These examples should not be construed tolimit the disclosure to the particular features or embodimentsdescribed.

EXAMPLES

There is no in vitro cell culture system to isolate and propagate NoV,impairing antigenic studies and vaccine development. From the 90s, ithas been known that the expression of VP1 results in the formation ofvirus-like-particles (VLPs) that have been shown to be morphologicallyand antigenically similar to the native virion (Jiang et al., J Virol66(11): 6527-32, 1992). VP1-VP2 VLPs vaccines are currently underevaluation (LoBue et al., Vaccine 24(24): 5220-34, 2006; Atmar et al., NEngl J Med 365(23): 2178-87, 2011; Wang et al., Vaccine 32(4): 445-52,2013; Willyard, Nat Med 19(9): 1076-7, 2013; Scotti and Rybicki, ExpertRev Vaccines 12(2): 211-24, 2013) but there is no vaccine yet available.To date, there is no therapy available for the prevention or treatmentof NoV diarrhea.

Crystallographic studies showed that NoVs bind carbohydrates of thehuman histo-blood group antigens (HBGAs) through the P2 protrudingdomain of VP1 (Cao et al., J Virol 81(11): 5949-57, 2007; Choi et al.,Proc Natl Acad Sci U.S. 105(27): 9175-80, 2008). A mechanism thatconsiders that this binding facilitates viral entry into the epithelialcells of the gastrointestinal tract had been proposed (Zhang et al.,PLoS One 8(7): e69379; 2013) It is thought that susceptibility to NoV inhumans is determined by allelic variation of HBGAs (Tan and Jiang,Trends Microbiol 19(8): 382-8 2011). The measurement of antibodies thatblock the interaction between VLPs and HBGA carbohydrates might serve asa surrogate neutralization test. Several sources of carbohydrates havebeen used in these surrogate neutralization tests: syntheticcarbohydrates, saliva, and pig gastric mucin (Harrington et al., J Virol76(23): 12335-4, 2002; LoBue et al., Vaccine 24(24): 5220-34, 2006). Thehemagglutination inhibition assay (IHA) using human red blood cell hasalso been used as a surrogate neutralization test (Hutson et al., JInfect Dis 185(9): 1335-7, 2002; Czako et al., Clin Vaccine Immunol19(2): 284-7, 2012) insert. A correlation has been reported between 1HAb titers and the susceptibility to infection (Czako et al., ClinVaccine Immunol 19(2): 284-7, 2012).

Because VLPs are antigenically similar to native virions (Jiang et al.,J Virol 66(11): 6527-32, 1992), conventional monoclonal antibodies(MAbs) against NoV have been developed after immunization of mice withVLPs from several NoV genotypes (Hardy et al. Virology 217(1): 252-61,1996; Yoda et al., Microbiol Immunol 44(11): 905-14, 2000; Yoda et al.,BMC Microbiol 1: 24, 2001; Almanza et al.,” J Clin Microbiol 46(12):3971-9, 2008). Several cross-reactive MAbs have been identified, andmost of them have been mapped to the S domain or the C-terminal regionof the P1 domain (Li et al. Virus Res 151(2): 142-7, 2009; Yoda et al.,BMC Microbiol 1: 24, 2001; Yoda et al., J Clin Microbiol 41(6): 2367-71,2003; Parker et al., J Virol 79(12): 7402-9, 2005; Batten et al.,Virology 356(1-2): 179-87, 2006; Oliver et al., J Clin Microbiol 44(3):992-8, 2006; Shiota et al., J Virol 81(22): 12298-306, 2007; Almanza etal., J Clin Microbiol 46(12): 3971-9, 2008; Parra et al., PLoS One 8(6):e67592, 2013). It has been reported that certain MAbs block theinteraction of VLPs with cells or synthetic HBGA (Lochridge et al., JGen Virol 86(Pt 10): 2799-806, 2005; Lindesmith et al., J Virol 85(1):231-42, 2011), and five HBGA-blocking sites have been mapped recently(de Rougemont et al., J Virol 85(9): 4057-70, 2011; Lindesmith et al.,PLoS Pathog 8(5): e1002705, 2012). Single chain antibodies (scFv) werealso constructed. Other non-conventional monoclonal antibodies have alsobeen developed against Norwalk virus (Chen et al., J Virol 87(17):9547-57, 2013). However, for an immunotherapy it is desirable to use aMAb derived from humans or chimpanzees or a molecule easy to humanize inorder to reduce a host immune response to the MAb during and after thetreatment to avoid the development of allergy and other hypersensitivityreaction.

A novel source of recombinant monoclonal antibodies derived from llamasis disclosed herein. IgG2 and IgG3 subtypes of the llamas lack the lightchain and are called heavy chain antibodies; the variable domain ofthese antibodies is called V_(H)H and is comprised of only onepolypeptide chain. The V_(H)H domain is a molecule of 15 kDa that is thesmallest known domain that occurs in mammals, with full antigen-bindingcapacity showing affinities comparable to conventional antibodies(Muyldermans, Annu Rev Biochem 82: 775-97, 2013).

Disclosed herein are recombinant monoclonal V_(H)H, specific for twodistinct NoV genogroups, GI.1 (Norwalk) and GII.4 (MD2004). In thesestudies, two llamas were immunized with GI.1 or GII.4 virus-likeparticles (VLPs). The surrogate neutralization assays indicated that theV_(H)Hs nanoantibodies were able to block the binding of VLPs todifferent sources of carbohydrates. V_(H)H antibodies were produceddirected to human NoV. The generated V_(H)H libraries represent a sourceof high quality reagents to be used for the antigenic characterizationof the virus and virus detection, and possess high potential forimmunoprophylaxis and therapeutic treatment of human NoV diarrhea.

Example 1 Materials and Methods

Expression and Purification of VLPs:

NoV VLPs containing VP1 and VP2 capsid proteins were expressed in abaculovirus system as described previously (Bok et al., J Virol 83(22):11890-901, 2009; Esseili et al., Appl Environ Microbiol 78(3): 786-94,2012; Green et al., J Clin Microbiol 35(7): 1909-14, 1997; Green, etal., J Clin Microbiol 31(8): 2185-91, 1993; Leite et al., Arch Virol141(5): 865-75, 1996; Lew et al. Virology 200(1): 319-25, 1994).Briefly, the ORF2 and ORF3 genes of several norovirus strains wereamplified by PCR and cloned into a pENTR plasmid (Invitrogen, Carlsbad,Calif.) to yield pENTRNoVVP1+VP2. Alternatively for some strains onlyVP1 VLPs were generated. Recombination of plasmid DNA with baculovirusDNA was performed using a Baculo direct kit (Invitrogen), and abaculovirus stock was obtained following transfection of therecombination product into Sf9 cells (serum-free adapted Sf9 cells;Invitrogen) as recommended by the manufacturer. The baculovirus stockwas used to infect Sf9 suspension cultures for VLP production. Culturemedium from baculovirus-infected cells was layered onto a 25% (wt/vol)sucrose cushion and subjected to centrifugation in an SW28 rotor at76,200 g for 4 h at 4° C. The resulting pellets were dissolved inphosphate-buffered saline (PBS), pH 7.4, and further purified through acesium chloride (CsCl) gradient by centrifugation in an SW55 rotor at218,400 g for 18 h at 15° C. The collected fractions (densities of 1.3g/ml) were dialyzed against PBS, and the protein concentration wasdetermined with a commercial Bradford assay kit (Pierce, Rockford,Ill.). The VLPs were purified using a combination of sucrose and CsClgradients and dialyzed in PBS overnight. The presence of VLPs wasconfirmed by electron microscopy.

For the different assays of this work VLPs of the following NoV strainswere included: Hu/NoV/GI.1/Norwalk/1968/U.S.,Hu/NoV/GI.1/P7-587/2007/Stromstad/Sweden;Hu/NoV/GI.3/Desert-Shield395/1990/U.S.; Hu/NoV/GII.4/MD2004/2004/U.S.,Hu/NoV/GII.4/MD145-12/1997/U.S.; Hu/NoV/GII.1/Hawaii/1971/U.S.;Hu/NoV/GII.2/Henryton/1971/U.S.; Hu/NoV/GII.3/Toronto24/1991/CA;Hu/NoV/GII.4/CHDC4871/1977/U.S.; Hu/NoV/GII.4/HS-191/2001/U.S.,Hu/NoV/GIV.1/SaintCloud624/1998/U.S., Hu/NoV/GII.3/Aus2001/2001/Aus,Hu/NoV/GII.3/Aus2007/2007/Aus, Hu/NoV/GII.3/Aus2008/2008/Aus,Hu/NoV/GII.3/CHDC2005/2005/U.S., Hu/NoV/GII.3/CHDC5261/1990/U.S.,Hu/NoV/GII.3/CHDC4031/1988/U.S.; Hu/NoV/GII.3/CHDC32/1976/U.S.,Hu/NoV/GII.7/DC119/U.S., Hu/NoV/GII.3/Maizuru/2000/JP,Hu/NoV/GII.14/M7/1999/U.S., Hu/NoV/GI.5/SzUG1/1997-99/JP,Hu/NoV/GI.6/Hesse/1997/GE, Hu/NoV/Snow Mountain/GII.2/U.S.,Hu/NoV/GII.4/RockvilleD1/2012/U.S. and Hu/GII.6/Bethesda/2012/U.S.

As negative control, VLPs of Vesiviruses V1415 and Mink calicivirus wereincluded. For the determination of the specificity of the V_(H)H againstthe different subdomains of VP1, the VLP NV S domain/MD2004 P domainchimera and the VLP MD2004 S domain/NV P domain chimera were generatedas previously described (Parra et al., Vaccine 30(24): 3580-6, 2012).Mutated VLPs used for epitope mapping were expressed and purified as theother VLPs used in this study.

Llama Immunization:

Two male llamas of one year of age were immunized by intramuscularinjection with 3 doses (day 0, 30 and 73) or 4 doses (day 0, 30, 73 and225) of vaccine containing around 300 μg/per dose of NoV VLP fromNorwalk strain (Hu/NoV/GI.1/Norwalk/1968/U.S.), or from MD2004 strain(Hu/NoV/GII.4/MD2004/2004/U.S.) respectively. For the first immunizationVLP's were emulsified in complete Freund's adjuvant. The followingimmunizations were formulated in incomplete Freund's adjuvant. Noadverse effects were observed after inoculation. Llama management,inoculation, and sample collection were conducted by trained personnelunder the supervision of a veterinarian and in accordance with approvedprotocols. Serum and blood samples were taken at days 0, 4 and 7 aftereach inoculation. The antibody responses to NoV in serum during the timecourse of immunization were monitored by ELISA. To evaluate the effectorB-cell response, an ELISPOT assay determining the number of NoV-specificantibody-secreting cells was performed at 4 and 7 days post eachinoculation.

NoV VLP ELISA:

ELISA was performed as disclosed previously (Bok et al., Proc Natl AcadSci U.S.A. 108(1): 325-30, 2011) with modifications for llama serum, asdescribed below: 96 flat bottom well polyvinyl microtiter plates(Maxisorp, NUNC, Denmark) were coated with 50 μl/well of NoV VLP'sovernight at 4° C. (Norwalk or MD2004 VLPs were used at a concentrationof 1 μg/ml in Carbonate Buffer pH 9.6). The plates were washed with0.05% Tween 20-PBS, and then were blocked with 200 μl of skim milk 5% inPBS for 1 hr, 37° C. After washing with 0.05% Tween 20-PBS, 50 μl ofeach serum sample four-fold dilution were added beginning with 1:50 in1% skim milk-PBS. Samples were run in duplicate wells and incubated at37° C. during 2 hours. The plates were washed with 0.05% Tween 20-PBSand a peroxidase-labeled anti-Llama IgG (Bethyl Labs, Inc., Montgomery,Calif.) at a 1:1,500 dilution in 1% skim milk-PBS was added to theplates, 50 μl/well. After 1 hour incubation at 37° C. the plates werewashed with 0.05% Tween₂₀-PBS and then the assay was developed withcommercial ABTS[2,2azinobis(3-ethylbenzthiazolinesulfonicacid)]/H₂O₂substrate: 100 μl/well. The absorbance at 405 nm was read in an ELISAreader (Multiskan EX, Thermo scientific, U.S.A.).

NoV VLP ELISPOT:

Antibody secreting cells (ASC) in the peripheral blood of the inoculatedllama was adapted from previous ELISPOT assays conducted for Rotavirus(Garaicoechea et al., J Virol 82(19): 9753-64, 2008). Briefly, 96-wellMaxisorp ELISA plates with flat bottom were coated with NoV VLPs dilutedin PBS (100 ng/well), overnight at 4° C. Suspensions of mononuclearcells derived from peripheral blood of the inoculated llamas were addedto quadruplicate wells in ten fold dilutions, starting with 1×10⁶cells/well (e.g., 1×10⁶, 1×10⁵, 1×10⁴, and 1×10³ cells per well). Aftercentrifugation at 500×g for 5 min, plates were incubated for 12 to 14 hat 37° C. in 5% CO₂. The plates were washed with PBS-0.05% Tween 20 toremove adherent cells, and the antibody spots were developed by adding aperoxidase-labeled goat anti-llama immunoglobulin G (IgG; H+L; BethylLabs, Inc., Montgomery, Calif.) at a 1/1,500 dilution for 2 h at 37° C.,and after washing the plate the assay was developed with 50 μl 1 of atetramethylbenzidine membrane peroxidase (TMB) substrate system (KLP,Maryland, U.S.A.).

V_(H)H Library Production:

Two V_(H)H libraries were developed from the blood of the two immunizedllamas. From the llama immunized with VLPs of NoV MD2004 (GII.4) 300 mlof blood were taken four days after the third dose and 300 ml of bloodfour days after the fourth dose. From the other llama immunized withVLPs of NoV Norwalk (GI.1), 450 ml of blood were taken 4 days after thethird dose. Mononuclear cells were extracted by Ficoll-Paque(Invitrogen, USA) gradient centrifugation, pelleted, frozen in liquidnitrogen, and then kept at 80° C. until the moment of use. The total RNAwas extracted by using a commercial RNA extraction kit (Nucleospin RNAL; Macherey Nagel), yielding 300 μg of RNA for the Norwalk llama and 420μg of RNA for the MD2004 llama. Subsequently, first-strand cDNA wassynthesized from the total RNA by using MMLV Reverse Transcriptase (RT)(Promega), with random primers (Invitrogen) according to themanufacturer instructions. For each 20 μl reaction, 10 μg of total RNAwas added. The V_(H)H-repertoire was PCR amplified from the total RNAfor each llama, using the four combinations of two forward primers andtwo reverse primers to amplify the different V_(H)H subfamilies ofshort-hinge and long-hinge. The primers contained the restriction sitesfor further cloning steps: V_(H)1b-SfiI (shf): 5′ GCT GGA TTG TTA TTACTC GCG GCC CAG CCG GCC ATG GCC CAG GT(GC) (AC)A(AG) CTG CAG SAG TCW GG3′ (SEQ ID NO: 35), Lam07-NotI (shr): 5′ GAT GGT GAT GAT GAT GTG CGG CCGCGC TGG GGT CTT CGC TGT GGT GCG 3′ (SEQ ID NO: 36), V_(H)6b-SfiI (lhf):5′ CGT GGA TTG TTA TTA TCT GCG GCC CAG CCG GCC ATG GCC GAT GTG CAG CTGCAG GCG TCT GG(AG) GGA GG 3′ (SEQ ID NO: 37), Lam08-NotI lhr: 5′ GAT GGTGAT GAT GAT GTG CGG CCG CTG GTT GTG GTT TTG GTG TCT TGG 3′ (SEQ ID NO:38). PCR amplification products were purified, restriction digested, andcloned into the SfiI and NotI sites of the phagemid vector pAO-Lib(Garaicoechea et al., J Virol 82(19): 9753-64, 2008), a modified versionof pHEN4 (Arbabi Ghahroudi et al., FEBS Lett 414(3): 521-6, 1997)carrying a long irrelevant sequence that is removed upon V_(H)Hinsertion in order to slow down the potential propagation of vectorwithout a V_(H)H insert. Ligated material was transformed intoEscherichia coli TG1 cells by electroporation. Colonies were harvestedby scraping in culture medium, washed and stored at −80° C. in LB mediumsupplemented with glycerol (50% final concentration).

Enrichment in V_(H)H of Interest:

The V_(H)H libraries were infected with M13K07 helper phages(Invitrogen), and phage particles expressing the V_(H)H repertoire wererescued and precipitated with polyethylene glycol as describedpreviously (Marks et al. J Mol Biol 222(3): 581-97, 1991). Enrichment inspecific binders was performed using the phage display technology by tworounds of in vitro selection, i.e. by the so-called “biopanning”.Briefly, Maxisorp immunoplates (NUNC) were coated overnight at 4° C.with NoV VLPs from Norwalk or from MD2004 strains (100 μg/well) incarbonate buffer pH 9.6. After a blocking step with skim milk 5% in PBS,phages from each library were added to the plates according to thedifferent biopanning strategies and incubated for 1 hour at roomtemperature. Two strategies of selection were performed, one in whichphages were incubated with the homologous VLPs, and the other strategythat had an additional step in which phages were incubated with theheterologous VLPs in order to subtract the cross reactive phages and theunbound phages were then incubated with the homologous VLPs. The secondstrategy was performed to enhance specificity and avoid selection ofcross reactive V_(H)Hs between both genogroups. After incubation, theplates were washed with PBS-Tween 0.05% and bound phage particles wereeluted with 100 mM triethylamine (pH 10.0) and immediately neutralizedwith 1M Tris (pH 7.4). The eluted phages were used to infectexponentially growing TG1 cells. After the second round of biopanning,individual colonies from 200 clones for NV and 200 clones for MD2004 NoVVLPs, were grown, and the corresponding V_(H)H clones were analyzed byphage ELISA for specificity to NoV GI.1 (Norwalk) and NoV GII.4(MD2004). Finally, a third biopanning strategy using polyvinyl ELISAplates coated with the VLPs diluted in PBS pH 7.4 was also conducted forthe GII.4 library.

Screening for GI.1 and GI.1 Specific V_(H)H Fragments by Phage ELISA:

Phages displaying the selected V_(H)H were produced by the individualTG1 Escherichia coli clones as previously described (Conrath et al.,Antimicrob Agents Chemother 45(10): 2807-12, 2001). ELISA plates werecoated overnight with 100 ng/well of NoV VLP of Norwalk strain or MD2004strain or blank. After the coating step, all plates were blocked with 5%skim milk in 0.5% Tween20-PBS. Phages from each clone were added towells coated with the different NoV VLPs and blank, and plates wereincubated at room temperature for 2 hours. The assay was developed usinga 1/5,000 dilution of a monoclonal antibody anti-M13p8(Amersham/Pharmacia Biotech) and then a 1/2,000 dilution of ananti-Mouse IgG conjugated with peroxidase (KPL), followed by theaddition of /ABTS/H₂O₂ as a substrate/chromogen reagent.

Expression and Purification of Recombinant V_(H)H:

V_(H)H cDNA of 11 clones that scored positive in phage ELISA for NoVVLPs of NV strain (GI.1) or MD2004 strain (GII.4) were subcloned usingthe restriction enzymes SfiI and NotI into the expression vector pHEN6(Conrath et al., Antimicrob Agents Chemother 45(10): 2807-12, 2001),which provides a pelB targeting sequence for the periplasm and aC-terminal His6 tag. Production of recombinant monovalent V_(H)H wasperformed in shaker flasks by growing cells in Terrific Brothsupplemented with ampicillin (Sambrook and Russell, Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory, 3rd edition, 2001). E.coli XL1-Blue cells were freshly transformed with the different plasmidconstructs and a culture from single colony for each V_(H)H was grown at37° C. and 250 rpm. V_(H)H expression was then induced with 1 mM IPTG[isopropyl-D-thiogalactopyranoside] for 16 h at 28° C. After the cellswere pelleted, the periplasmic proteins were extracted by osmotic shock(Skerra and Pluckthun Science 240(4855): 1038-41, 1988). The V_(H)H werepurified from this periplasmic extract by using a High-Trap HPNi-chelating column (Amersham Biosciences).

The V_(H)H nucleotide sequences of the obtained V_(H)H clones werealigned by ClustalW with Mega 6.06 and the alignment was edited withBioEdit.

NoV VLPs Detection by V_(H)H in ELISA:

Briefly, 96 well U bottom polyvinyl plates Dynatech (Nunc, Thermo,EEUU), were coated overnight (ON) at 4° C. with 100 ng of purifiedVLPs/well diluted in 50 μl of PBS pH 7.4. Wells incubated with PBS alonewere used as a negative control for V_(H)H binding. After washing theplates with PBS pH 7.4 0.1% Tween20, they were blocked with 200 μl ofskim milk 5% in PBS pH 7.4 0.1% Tween20, for 1 hour at 37° C. Then 50μl/well with the corresponding V_(H)H dilution in PBS pH 7.4 5% skimmilk were added and the plates were incubated for 2 hours at roomtemperature. After washing the plates, 50 μl of anti-V_(H)H serum madein rabbit and diluted 1:8,000 in PBS 5% skim milk, were added to eachwell and the plates were incubated for 1 hour at room temperature. Theplates were washed with 0.05% Tween 20-PBS and the binding of antibodiesto the VLP antigen was detected with horseradish peroxidase(HRP)-conjugated goat anti-rabbit IgG (KPL, Gaithersburg, Md.), 1:2,000dilution in 1% skim milk-PBS, 50 μl/well. After 1 hour of incubation at37° C., the plates were washed and the assay was developed with thechromogen system ABTS/H₂O₂. Absorbance at 405 nm was measured. The cutoff was defined as twice the absorbance obtained in the blank wells. Forthe determination of the V_(H)Hs detection limit for GI.1 (Norwalk andNorovirus 2007) and GII.4 (MD2004 and MD145) VLPs, serial ten-folddilutions were tested from 500 ng/well to 0.005 ng/well of V_(H)H. Totest the ability of each V_(H)H to recognize a panel of VLPs fromdifferent NoV strains, the fixed amount of 20 ng of V_(H)H/well wasselected.

To evaluate the specificity of the V_(H)H against the different domainsof VP1, the VLP NV S domain/MD2004 P domain chimera and the VLP MD2004 Sdomain/NV P domain chimera previously described were used (Parra et al.,J Virol 86(13): 7414-26, 2012). The V_(H)H were tested in this assay atthe fixed concentration of 20 ng/50 μl per well.

Western Blot Analysis:

The reactivity of each V_(H)H with the MD2004 VLPs or with the NorwalkVLPs was analyzed by Western blot. For this assay, 1.5 μg of VLPs weremixed with NOVEX® 2×Tris-Glycine SDS loading buffer (Invitrogen), andafter boiling during 5 min at 95° C., the samples were analyzed withpolyacrylamide gel electrophoresis (SDS-PAGE) NUPAGE® Novex 4-12%(Invitrogen). The proteins were blotted onto a nitrocellulose membraneusing the IBLOT® Dry Blotting System (Invitrogen). The membranes wereblocked with PBS 5% skim milk for 2 h at RT. Each V_(H)H (5 μg/ml) wasadsorbed overnight at 4° C. and the binding was detected with rabbitanti V_(H)H antiserum 1:1,000 1 hour at room temperature and thenAlkaline Phosphatase-conjugated goat anti-rabbit IgG (1:2,000, 1 hour atroom temperature) and the NBT-BCIP Chromogen system.

Blocking of NoV VLP-Binding to Synthetic HBGA by V_(H)Hs:

Neutravidin coated plates (PIERCE®) were used for this assay followingthe manufacturer instructions. Plates were coated with biotinylatedcarbohydrates H1 (for NV) or H3 (for MD145) 1 μg/well. A total of 150 ngof VLPs diluted in 100 μl of buffer were pre-incubated with 400, 200,100, 50, 25 and 0 ng of V_(H)H for 1 hour at room temperature. The 150ng of the pre-incubated VLPs were added to each well of thecarbohydrate-coated plates and incubated for 1 h. The binding ofcaptured VLPs was determined by incubation with guinea pig hyperimmuneserum (1:10,000 dilution), followed by incubation with HRP-conjugatedgoat anti-guinea pig immunoglobulin G (1:2,000 dilution; KPL) and theperoxidase substrate ABTS (KPL). All incubations were performed at roomtemperature. Absorbance at 405 nm was measured. The percent controlbinding was defined as the binding level in the presence of antibodypretreatment divided by the binding level in the absence of antibodypretreatment multiplied by 100. Mean % control binding present theresults of two replicates for each dilution tested. An antibody wasdesignated as a “blockade” antibody for a VLP if at least 50% of controlbinding (EC50) was inhibited by 2 μg/ml antibody or less. Blockade datawere fitted and EC50 values calculated using sigmoidal dose responseanalysis of non-linear data in GraphPad Prism 5 (available on theinternet, graphpad.com).

As positive controls for the blocking of the binding of the VLPs, themonoclonal antibody D8 anti-Norwalk and the monoclonal antibody C9anti-MD145 were included.

VLP-Pig Gastric Mucin Ligand-Binding Antibody Blockade Assays:

Pig Gastric Mucin Type III (PGM) (Sigma Chemicals) was used as asubstrate for NoV VLP antibody-blockade assay as previously described.Briefly, PGM was resuspended in PBS at 5 mg/ml and coated onto 96 well Ubottom polyvinyl plates Dynatech (Nunc, Thermo, USA) at 10 μg/ml in PBSfor 4 hours. Plates were then blocked ON at 4° C. in 5% dry milk in0.05% Tween₂₀-PBS. Norwalk and MD 2004VLPs (0.5 mg/ml) were pretreatedwith decreasing concentrations of each V_(H)H (2-fold dilutions from 8)μg/ml to 0.125 ug/ml) for 1 hour. One hundred μl of VLPs-V_(H)H mix weretransferred to the PGM coated plates and incubated for 1 hour. BoundVLPs were detected by specific hyperimmune sera made in guinea pig,followed by anti-guinea pig IgG-HRP (KLP). ABTS/H₂O₂ was used aschromogen/substrate solution. All incubations were performed at roomtemperature and absorbance at 405 nm was measured. Mean % controlbinding, EC50 and criteria for determination of a blockade V_(H)H werecalculated as described above.

VLP-Saliva Ligand-Binding Antibody Blockade Assays:

For this assay saliva positive for Ly antigen collected from a secretorindividual was used. Saliva was boiled (95° C.) for 10 minutesimmediately after collection, centrifuged for 5 minutes at 13000×g andthe supernatants stored at −20° C. until used. ELISA 96 well U bottompolyvinyl plates Dynatech (Nunc, Thermo, EEUU) were coated with 100μl/well of a 1/400 saliva dilution in 50 μM carbonate-bicarbonatebuffer, pH 9.6. The plate was incubated overnight at 37° C. in a wetatmosphere. In parallel, serial two-fold dilutions of V_(H)H startingfrom 8 ug/ml were mixed with 1.5 ug/ml of NoV VLP Norwalk and MD145 andincubated for 1h at 37° C. After 6 washes and a blocking step with 5%dry milk/0.05% Tween₂₀-PBS, 50 ul of each V_(H)H-VLP mixture wastransferred to the reaction plate, in duplicate. The assay was developedusing a specific polyclonal antiserum made in guinea pig (1:10,000)followed by a peroxidase labeled anti-guinea pig IgG (1:2,000) andABTS/H₂O₂ as substrate chromogen (KPL), 100 μl per well. Absorbance at405 nm was measured. Mean % control binding, EC50 and criteria fordetermination of a blockade V_(H)H were calculated as described above.

Hemagglutination Inhibition Assay (HAI):

This assay was performed using red blood cells (RBC) that had theproperty of hemagglutinating the different VLPs tested: MD2004, MD145,NV and P7-587. Several human blood samples were tested forhemagglutination property to the four selected VLPs. Finally 0 Rh-bloodsamples were selected to perform the assay with GI.1 VLPs and B Rh+ wasselected for hemagglutination of GII.4 VLPs. The samples, RBC andbuffers were prepared as described elsewhere (Hutson et al., J InfectDis 185(9): 1335-7, 2002). The working solution of RBC was preparedadding 0.75 ml of RBC pellet to 100 ml of physiologic saline solutionpH6.2. The V_(H)H samples were diluted in PBS-physiologic salinesolution pH 5.5 from 6.25 μg/25 μl to 0.0025 μg/25 μl in two fold serialdilutions and 25 μl of each dilution were added in duplicates to eachwell. Four to eight hemagglutination units (HU) of VLPs were added toeach sample dilution and the plates were incubated for 1 hour at roomtemperature. Then, 50 μl of the RBC dilution 0.75% in physiologic salinesolution pH 6.2 were added to each well and the plates were incubatedfor 2 hours at 4° C. and finally the presence or absence ofhemagglutination was observed. The HAI titer of each V_(H)H was definedas the lowest antibody concentration that completely prevented NoVVLP-induced hemagglutination by visualization.

Blocking Between V_(H)Hs and Llama Hyperimmune Serum for NoV VLPBinding:

Briefly, 96-well polyvinyl microtiter plates (Thermo) were coated with12.5 ng/well of VLPs (from Norwalk or MD2004 NoV strains) and incubatedovernight at 4° C. Wells were washed with 0.05% Tween₂₀-PBS and blockedwith 5% dry milk-PBS for 1 h at RT.

In the first assay, llama hyperimmune serum specific for thecorresponding VLP, was added to each well diluted 1/500 and incubatedfor 1 hour at room temperature. After washing the plates, 6.25 ng ofeach V_(H)H were added to the wells in duplicates. The same assaywithout blocking with the hyperimmune antisera was used as control. Thenthe plates were washed and incubated with anti His MAb (Qiagen) 10ng/well and HRP-conjugated anti-mouse immunoglobulin G (1/2,000) (KPL).The signal was developed with ABTS/H₂O₂ and the absorbance at 405 nm wasmeasured.

In another assay, each V_(H)H or a pool of all V_(H)Hs in aconcentration of 250 μg/ml were added to each well and incubated for 1hour at room temperature, control wells without blocking V_(H)Hs wereincluded. After washing the plates, two fold serial dilutions of thecorresponding llama hyperimmune serum from 1/1,000 to 1/2,000,000 wasadded in duplicates. The assay was developed with HRP-conjugatedanti-llama IgG (1/2,000) (Bethyl). The signal was developed withABTS/H₂O₂ and the absorbance at 405 nm was measured.

Immunofluorescence Assay to Evaluate Competition Between the DifferentV_(H)Hs:

Vero cells were plated in 96-well plates at 50,000 cells/well andincubated for 24 h at 37° C., 5% CO₂. The cells were then infected witha modified vaccinia virus expressing bacteriophage T7 RNA polymerase(MVA-T7) at a multiplicity of infection (MOI) of 5 PFU/cell for 1 h at37° C., 5% CO₂. After infection, cells were transfected with 400 ng/wellof each DNA construct expressing Norwalk or MD2004 VP1 in the presenceof Lipofectamine 2000 (Invitrogen) following the manufacturer'srecommendations. Transfected cells were incubated for 24 h and thenfixed with cold methanol for 10 min. The plates were blocked using 10%normal goat serum (KPL), ON at 4° C. Fifty microliter containing 10μg/well of each unlabeled V_(H)H and 1 μl of ALEXA FLUOR® 568 (MolecularProbes-Invitrogen, Carlsbad, Calif.) labeled V_(H)H (1 mg/ml) was addedto the fixed cells in duplicates and incubated for 2 h at roomtemperature.

Epitope Mapping of the V_(H)H that Recognized a Linear Epitope Using aPeptide Library:

For this assay, a peptidic library of 67 peptides corresponding to the Pdomain of the VP1 amino acid sequence from NoV Toronto strain wassynthetized. The peptides were 17 amino acids long, with 5 amino acidsoverlapping with the previous and the next peptide of the sequence.Neutravidin coated plates (PIERCE®) were used for this assay. The plateswere coated with each biotinylated peptide in duplicates (2 μl ofpeptide/100 μl of PBS 0.1% BSA per well) overnight at 4° C. Then, afterwashing the plates, 100 ng/well of 7.5 V_(H)H were added and the plateswere incubated for 2 hours at room temperature. The plates were washedand incubated with rabbit anti-V_(H)H serum (1/8,000) and HRP-conjugatedgoat anti-rabbit immunoglobulin G (1:2,000) (KPL). The signal wasdeveloped with ABTS/H₂O₂.

Site-Directed Mutagenesis for Epitope Mapping of V_(H)H 16.

The pCI-MD2004 vector described previously (Parra et al., J Virol86(13): 7414-26, 2012) was mutated using a Quick-change site-directedmutagenesis kit (Stratagene) and complementary forward and reverseprimers which carried the nucleotide mutations. The restriction enzymeDpnI (10 U/μ1) was used to digest the parental DNA. Each of the mutatedproducts was transformed into Epicurian Coli XL1-Blue supercompetentcells (Stratagene). Transformed cells were grown overnight in LB plateswith carbenicillin (50 μg/ml), and individual colonies were used forplasmid amplification. The resulting plasmids were subjected to sequenceanalysis to verify the entire VP1 coding region and confirm the presenceof the introduced mutations. The mutation sites were selected accordingto the different surface amino acids between MD2004 and MD145 strains.The ability of V_(H)H 16 to recognize the mutated VP1 was tested byimmunofluorescence and by ELISA.

Briefly, MD2004 mutant VP1 were expressed in Vero cells as describedabove and after the incubation with V_(H)H 16 the assay was developedwith rabbit anti-V_(H)H polyclonal serum and goat anti-rabbit IgG(H+L)conjugated with Alexa Fluor 488 (Molecular Probes-Invitrogen, Carlsbad,Calif.). The plate was observed under the immunofluorescence microscopy.A V_(H)H that detected MD145 strain was included as positive control.

MD2004 mutant VLPs were expressed and purified as previously described(Parra et al., J Virol 86(13): 7414-26, 2012) and detected by ELISA asdescribed above.

Bioinformatics Analyses:

Nucleotide sequences from the prototype strains were downloaded fromGENBANK® and aligned by using the translated amino acid sequences.Phylogenetic trees were constructed using the Kimura two-parameter modelas a nucleotide substitution model and a neighbor-joining (NJ) algorithmas implemented in MEGA v4.0 (55). The solved structure of the P domainof VA387 virus (GII.4) in complex with carbohydrate (Protein Data Bank[PDB] accession number 2OBT) was used to identify the residues involvedin binding with MAbs and was visualized by using MacPyMol (DeLanoScientific LLC).

Example 2 Llama Immunization

The antibody (Ab) response to immunization is depicted in FIG. 1. Bothllamas were seronegative for Ab to human NoV GI.1 and GII.4 at thebeginning of the immunization. After 14 days post the first dose bothllamas developed strong antibody responses to the homologous VLP.Cross-reactive antibody responses were also detected. The llamaimmunized with Norwalk GI.1 NoV VLPs developed high ELISA Ab titerreaching a plateau at 1:100,000 serum dilution. A cross reactiveantibody (Ab) response to MD2004 GII.4 VLPs of lower magnitude, reachinga plateau at 1:10,000 serum dilution was also observed (FIG. 1a ).

The llama immunized with MD2004 GII.4 NoV VLPs developed a strong Abresponse by ELISA to the homologous antigen reaching a plateau at1:800,000 serum dilution. This llama also showed seroconversion toNorwalk GI.1 VLPs but of lower magnitude reaching a plateau at 1:1,000serum dilution (FIG. 1b ).

Antibody secreting cell (ASC) responses were successfully assessed forNorwalk and MD2004 NoV VLPs. These results showed the presence of highnumbers of ASC to the homologous VLP and around of 1-2% of ASC withcross reactivity (FIGS. 1a and b ).

After confirming optimal Ab titer and ASC responses, both llamasreceived a third immunization and were bled 4 days after the third dose.Additionally, the llama immunized with MD2004 VLPs received a fourthdose of antigen and was also bled four days later. A total of 450 ml ofblood was obtained from the llama immunized with Norwalk NoV VLPsyielding 5.91×10⁸ mononuclear cells, while 600 ml of blood extractedfrom the llama immunized with MD2004 NoV VLP yielded 6.6×10⁸ mononuclearcells. From the processed RNA, two V_(H)H phage display librariescontaining 4.2×10⁸ clones for Norwalk NoV and 1.0×10⁸ clones for MD2004NoV were generated. Both V_(H)H libraries possessed optimal size (in theorder of 10⁸) according to the literature (Muyldermans, Annu Rev Biochem82: 775-97, 2013).

Example 3 Phage Display Selection of V_(H)H Specific to Norwalk orMD2004 NoV Strains

To select phages displaying V_(H)H specific for each NoV genogroup, tworounds of in vitro selection (biopanning) were performed using thehomologous NoV VLPs as antigens. Furthermore, with the aim of obtaininghighly specific V_(H)Hs to the immunization antigen, two round of anextra biopanning strategy were performed. The phages from each librarywere pre-incubated with the heterologous antigen to subtract the crossreactive clones and the unbound phages were then incubated with thehomologous antigen. After the second round of biopanning, 96 clones foreach sub-library (a total of 384 clones) were analyzed by phage ELISA.

Ninety out of 96 clones from the V_(H)H library specific for Norwalkrecognized Norwalk VLPs, when the library was enriched with thehomologous VLP. In the second biopanning strategy, when cross reactiveclones were previously subtracted with MD2004 VLPs and the library wasthen enriched in Norwalk specific clones, 32/96 phages recognizedNorwalk VLP by phage ELISA.

For the MD2004 specific library, biopanned with the homologous antigen,31/96 phages recognized MD2004 VLPs in phage ELISA. When a previous stepof subtraction of cross reactive phages with Norwalk VLPs was performed,44/96 clones recognized MD2004 VLPs. For all the tested phages, noreaction was observed for the blank plates nor for the plates coatedwith the VLP not used in the biopanning.

From the total positive clones obtained in each initial biopanningcondition, the ones with the highest positive signal in phage ELISA wereselected, subcloned, expressed and purified.

From these clones, 16 V_(H)H and 2 VH with different amino acidsequences that were successfully subcloned, expressed in the pHEN6expression vector and purified by his-tag, were chosen for furthercharacterization experiments, 10 V_(H)H derived from the Norwalk librarywhile 6 V_(H)Hs and 2 VHs derived from the MD2004 library (Table 1).

TABLE 1 Selection of V_(H)H for characterization Antigen Antigen Antigendetected of of by Phage Ab vaccination selection ELISA Clone typeNorwalk Norwalk Norwalk 6.3 V_(H)H 7.3 8.1 9.8 10.4 11.2 12.2 14.5Norwalk Norwalk 13.2 V_(H)H (-MD2004) H1 MD2004 MD2004 MD2004 1.1 V_(H)H2.1 3.2 4.1 5.4 7.5 MD2004 MD2004 16 VH (-Norwalk) 19

The third biopanning strategy using polyvinyl plates coated with GII.4VLPs in PBS pH 7.4 was conducted in a pH that improve the stability ofthe VLP in order to select V_(H)Hs to other native epitopes. From thiscondition 13 new clones were obtained.

The aligned sequences of the obtained V_(H)H clones are shown in FIG. 8and the CDR3 of the V_(H)Hs are detailed in Table 5.

TABLE 5 CDR3 sequences of the V_(H)Hs NoV SEQ VHH Amino acid genogroupID name sequences of the CDR3* specificity NO:*  3.2 NLKRRDLQARFGGY GII1  4.1 NLKRRDLQSRFGGY GII 2  5.4 NLKRRDLQARFGGY GII 3 P10 NLKRRDLQARFGGYGII 4 19.1 AKPRDFWYSPEFDF GII 5 P3 AKGVYGSRRSADFGS GII 6 P4AKGVYGSRRSADFGS GII 7 P5 AKGVYGSRRSADFGW GII 8  7.5 NANFQIHRSGADYVRNYGII 9 P15 NANLQIHRDSSGDVRNV GII 10 P2 NANLQISRSEDGAYVVRNY GII 11 P8NANLQFYRGGGSDVKNY GII 12  2.1 AAAEFFSSGDPLPGMDY GII 13 P12AAAEFLPTQRSPREYDY GII 14 P14 AASRRFWTAALNGADYPY GII 15 P13 NARDWSDGFDEYGII 16 P9 ASGPRANASIRRSGYNY GII 17  1.1 TASEFLLHPPPPNQKYDY GII 18 P1AARSRPAISTRRPDYFA GII 19 P7 AARRRVFSRAAAAYNY GII 20 16.1 SRGVSGE GII 21 6.3 YALIQTASTTWYRQY GI 22  7.3 NANLGALLDY GI 23  8.1 KRVRDVIGRPEL GI 24 9.8 AAEVHPGDYGLTYMQSQYEYDY GI 25 10.4 KVDSYTYGTDI GI 26 11.2KADGRRYSLNEY GI 27 12.2 NIYYGGDYYYTGVKPNP GI 28 13.1 AASKIRNDIYLNDYTWYQYGI 29 14.5 AAHHITPTGSYYYSEPLPVDMVYDY GI 30 *The sequences presented are:a) amino acids 96-109 of SEQ ID NO: 1; b) amino acids 96-109 of SEQ IDNO: 2; c) amino acids 96-109 of SEQ ID NO: 3; d) amino acids 96-109 ofSEQ ID NO: 4; e) amino acids 97-110 of SEQ ID NO: 5; f) amino acids97-111 of SEQ ID NO: 6; g) amino acids 97-111 of SEQ ID NO: 7; h) aminoacids 97-111 of SEQ ID NO: 8; i) amino acids 96-112 of SEQ ID NO: 9; j)amino acids 96-112 of SEQ ID NO: 10; k) amino acids 96-114 of SEQ ID NO:11; l) amino acids 96-112 of SEQ ID NO: 12; m) amino acids 97-113 of SEQID NO: 13; n) amino acids 97-113 of SEQ ID NO: 14; o) amino acids 97-114of SEQ ID NO: 15; p) amino acids 96-107 of SEQ ID NO: 16; q) amino acids97-113 of SEQ ID NO: 17; r) amino acids 97-114 of SEQ ID NO: 18; s)amino acids 97-113 of SEQ ID NO: 19; t) amino acids 96-111 of SEQ ID NO:20; u) amino acids 96-102 of SEQ ID NO: 21; v) amino acids 96-110 of SEQID NO: 22; w) amino acids 95-104 of SEQ ID NO: 23; x) amino acids 96-107of SEQ ID NO: 24; y) amino acids 100-110 of SEQ ID NO: 25; z) aminoacids 96-117 of SEQ ID NO: 26; aa) amino acids 97-108 of SEQ ID NO: 27;bb) amino acids 96-112 of SEQ ID NO: 28; cc) amino acids 97-115 of SEQID NO: 29; dd) amino acids 97-121 of SEQ ID NO: 30.

Example 4 V_(H)H Domain Specificity on the VP1 Protein and Western BlotAnalysis

All the V_(H)Hs selected were directed to the P domain of VP1 (Table 2).

TABLE 2 Specificity of the V_(H)Hs to the P or S domain of VP1 VHH GI.1specific VHH GII.4 specific VLP 6.3 7.3 8.1 9.8 10.4 11.2 12.2 13.1 14.5H1 1 2.1 3.2 4.1 5.4 7.5 16 19 ChimMD2004/(S)-NV(P) + + + + + + + + + +Chim NV(S)/MD2004(P) + + + + + + + + Norwalk 1968 + + + + + + + + + +MD2004 + + + + + + + + Recognized Domain P P P P P P P P P P P P P P P PP P

All V_(H)Hs, except clone 7.5, failed to recognize the NoV VLPs by WB,suggesting that they are directed to conformational epitopes of the Pdomain. In contrast clone 7.5 was able to recognize the VP1 of MD2004strain (homologous) by WB as well as the VP1 of GII NoVs strainsbelonging to different genotypes (FIG. 9A-9B).

Example 5 V_(H)H Recognition of NoV VLPs from Different Years andGenotypes by ELISA

The purified V_(H)H were tested by ELISA against the NoV VLPs used inthe immunization and biopanning and also with a panel of 26 VLPsrepresenting different genogroups/genotypes of NoV (Table 3).

TABLE 3 Capacity of the V_(H)Hs of recognizing VLPs of differentgenotypes by ELISA GI.1-specific VHH Norovirusstrain Year Genotype 6.37.3 8.1 9.8 10.4 11.2 12.2 13.1 14.5 H1 Norwalk 1968 1968 GI.1 0.50 0.501.00 0.50 0.50 1.00 1.00 2.00 0.50 1.00 P7-587 2007 GI.1 0.50 0.50 1.001.00 0.50 1.00 1.00 − 0.50 − Desert-Shield395 1990 GI.3 − 0.50 − − − − −− − − SzUG1 1997-99 GI.5 − − − − − − − − − − Hesse 1997 GI.6 − − − − − −− − − − Hawaii 1971 GII.1 − − − − − − − − − − Snow Mountain 1976 GII.2 −− − − − − − − − − Henryton 1971 GII.2 − − − − − − − − − − Toronto 241991 GII.3 − − − − − − − − − − CHDC2005 2005 GII.3 − − − − − − − − − −CHDC5261 1990 GII.3 − − − − − − − − − − CHDC4031 1988 GII.3 − − − − − −− − − − Maizuru2000 2000 GII.3 − − − − − − − − − − Aus2001 2001 GII.3 −− − − − − − − − − Aus2007 2007 GII.3 − − − − − − − − − − Aus 2008 2008GII.3 − − − − − − − − − − CHDC32 1976 GII.3 − − − − − − − − − − CHDC48711977 GII.4 − − − − − − − − − − Rockville 2012 GII.4 − − − − − − − − − −MD2004 2004 GII.4 − − − − − − − − − − MD145 1997 GII.4 − − − − − − − − −− HS191 2001 GII.4 − − − − − − − − − − Bethesda 2012 GII.6 − − − − − − −− − − DC119 1978 GII.7 − − − − − − − − − − M7 1999 GII.14 − − − − − − −− − − St. Cloud 624 1998 GIV.1 − − − − − − − − − − GII.4-specific VHHNorovirusstrain Year Genotype 1 2.1 3.2 4.1 5.4 7.5 16 19 Norwalk 19681968 GI.1 − − − − − − − − P7-587 2007 GI.1 − − − − − − − −Desert-Shield395 1990 GI.3 − − − − − − − − SzUG1 1997-99 GI.5 − − − − −− − − Hesse 1997 GI.6 − − − − − − − − Hawaii 1971 GII.1 − − − 1.00 1.001.00 − 1.00 Snow Mountain 1976 GII.2 − − − 2.00 2.00 2.00 − 2.00Henryton 1971 GII.2 − − − 1.00 2.00 1.00 − 2.00 Toronto 24 1991 GII.3 −− − + + + − + CHDC2005 2005 GII.3 − − − + + + − + CHDC5261 1990 GII.3 −− − + + + − + CHDC4031 1988 GII.3 − − − 2.00 0.50 1.00 − 0.50Maizuru2000 2000 GII.3 − − − + + + − + Aus2001 2001 GII.3 − − − + + +− + Aus2007 2007 GII.3 − − − + + + − + Aus 2008 2008 GII.3 − − − + + +− + CHDC32 1976 GII.3 − − − + + + − + CHDC4871 1977 GII.4 0.25 − 0.500.50 0.50 2.00 − 1.00 Rockville 2012 GII.4 0.25 0.25 0.50 1.00 0.50 0.50− 2.00 MD2004 2004 GII.4 0.50 0.50 2.00 2.00 1.00 0.50 0.50 2.00 MD1451997 GII.4 0.50 8.00 1.00 2.00 4.00 0.50 − 4.00 HS191 2001 GII.4 1.000.50 0.50 0.50 0.25 2.00 − 1.00 Bethesda 2012 GII.6 − − 0.50 2.00 1.001.00 − 2.00 DC119 1978 GII.7 − − − 0.50 0.50 1.00 − 1.00 M7 1999 GII.14− − − − − 1.00 − − St. Cloud 624 1998 GIV.1 − − − − − − − −

Numbers detailed in the table represent the minimum concentration ofV_(H)H (ng/well) able to recognize 100 ng of purified VLPs by ELISA.

The ten V_(H)Hs obtained from the Norwalk library were able to reactwith the homologous GI.1 VLP. All clones except 13.1 and H1 were able todetect P7-587GI.1 strain. Only clone 7.3 was able to recognize theDesert Shield 395 strain that belongs to GI.3 genotype. None of theseV_(H)H reacted with GI.5 and GI.6 strains or with GII and GIV NoVs(Table 3).

The eight V_(H)Hs derived from the GII.4 MD2004 library were able todetect the homologous VLP with high affinity by ELISA. These V_(H)Hsdisplayed a great variety of recongnition patterns. Clone 16 reactedspecifically with GII.4 MD2004 strain. Clone 2.1 detected with highaffinity 3 out of 5 GII.4 strains, clone 1 reacted with all GII.4strains, clone 3.2 reacted with GII.4 strains and also detected GII.6strain. Clones 4.1, 5.4 and 19 were able to react with GII NoVs strainbelonging to genotypes 1, 2, 3, 6 and 7. Finally, the clone 7.5,directed to a linear epitope, recognized all the GII VLPs tested byELISA (Table 3).

The clone 13 and H1, specific for Norwalk and clone 16 specific forMD2004 (Table 3)—the antigens used for llama immunization-were selectedfrom subtractive biopanning strategies (Table 1). The 13 clones obtainedin the third biopanning strategy were able to recognized GII.4 MD 2004by ELISA with high affinity.

Using this panel of recombinant NoV VLPs covering a broad range ofgenotypes within both genogroups, the V_(H)H tested were shown to beeither genogroup specific, genotype specific, or strain specific. Thedifferent patterns of cross-reactivity were similar in nature to theobserved behavior of conventional monoclonal antibodies (Li et al.,Virus Res 151(2): 142-7, 2010; Yoda et al., BMC Microbiol 1: 24, 2001;Yoda et al., J Clin Microbiol 41(6): 2367-71, 2003; Parker et al., JVirol 79(12): 7402-9, 2005; Batten et al., Virology 356(1-2): 179-87,2006; Oliver et al., J Clin Microbiol 44(3): 992-8, 2006; Shiota et al.,J Virol 81(22): 12298-306, 2007; Almanza et al., J Clin Microbiol46(12): 3971-9, 2008; and Parra et al., Vaccine 30(24): 3580-6, 2012).

Example 6 Surrogate Assays of Virus Neutralization: HBGA, Pig GastrinMucin, Saliva Blockade Assays and Hemagglutination Inhibition Test

In order to study if the selected V_(H)H possess the property toneutralize NoV infection, a panel of neutralizing surrogate assays wereconducted and the summary of each V_(H)H behavior is summarized in Table4 and depicted in FIGS. 2 and 3.

TABLE 4 V_(H)H behavior in surrogate neutralizing assays: summaryresults EC50 HAI titer (μg/ml) (μg/ml) HBGA PGM Saliva 0 Rh − RBC H1type III Ly+ Norovirus V_(H)H Norwalk Norwalk Norwalk Norwalk 2007 GI.1VLP VLP VLP VLP VLP 6.3 1.006 0.661 2.035 0.19 0.05 7.3 — — — 0.39 0.058.1 — 4.139 — — 0.78 9.8 — 1.144 1.718 — 3.12 10.4 — 0.667 1.136 0.1 0.05 11.2 — 6.856 — — — 12.2 — 2.594 — — 0.8  13.1 2.032 1.289 2.2670.19 — 14.5 ND 0.351 1.563 0.78 0.05 H1 ND 0.275 2.637 0.19 — EC50(μg/ml) HAI titer HBGA PGM Saliva (μg/ml) H3 type III Ly+ B Rh + RBCV_(H)H MD145 MD2004 MD145 MD2004 MD145 GII.4 VLP VLP VLP VLP VLP 1 0.85 0.341 2.008 — — 2.1 — — — — — 3.2 0.641 2.296 0.917 0.19 0.19 4.1 0.7122.909 1.379 0.19 0.39 5.4 0.718 2.023 1.268 0.39 0.39 7.5 — — — — 0.7816 — 0.767 — — — 19 — 0.583 1.296 0.39 0.39 Positive values according tothe established cut off are indicated in bold. For blockade assay cutoff value is 2 μg/ml and for HAI assay the cut off value is 0.39 μg/ml.Seven out of the 10 V_(H)H specific to GI NoVs showed a blockadeproperty by some of the surrogate neutralization assays. The bestperformances were observed with clone 6.3 that was able to interfereGI.1 VLPs attachment to the carbohydrates in all the blockade assays andalso showed HAI properties. V_(H)H 10.4 also represents a good option,since it was able to inhibit both Norwalk and P7-587 VLP binding tocarbohydrates of the PGM, saliva and human RBC. Finally, clones 13.1 andH1 were effective but only specifically for Norwalk strain.

Regarding the V_(H)H derived from the GII.4 MD2004 library, clones 3.2,4.1 and 5.4 showed good EC₅₀ values to inhibit VLP attachment to H3carbohydrate, saliva and showed HAI, but higher amounts of V_(H)Hs wereneeded to interfere the attachment of the VLPs to the PGM (2 μg/ml orhigher).

Clone 1 was able to interfere with the VLP binding to syntheticcarbohydrate H3, PGM and human saliva, but failed to induce HI. Clone19, was able to inhibit VLP attachment to PGM, saliva and showed HAIproperties. Clone 16, was able to inhibit VLP attachment to PGM with agood EC50, but failed to block carbohydrate binding and did not show HAIactivity. Finally, V_(H)H 2.1 and 7.5 did not show blocking activity.

The results of cross-reactivity obtained by ELISA, together with theresults obtained in the neutralization surrogate assays utilizingdifferent sources of carbohydrates (NoV VLP-HBGA, PGM and salivablockade assays; and human RBC hemagglutination inhibition assays), allstrongly suggest that some of the obtained V_(H)H possess broadlypotential viral neutralizing activity, and that the antibodies that canbe utilized as a prophylactic or therapeutic intervention.

The clones directed toward GI.1 NoV did not display cross reactivity tothe other GI genotypes tested with the exception of the V_(H)H 7.3,which also recognized GI.3 Desert Shield strain by ELISA. Within the GIspecific V_(H)Hs, the clones 6.3, 10.4 and 14.5 all showed the bestblockade performance within the different carbohydrate sources. Thecalculated EC50 values for these V_(H)Hs ranged between 0.275 and 2.0μg/ml, all comparable to the EC50 values of the scFv derived fromimmunized chimpanzees (from 0.3 to 1.5 μg/ml) (Chen et al., J Virol87(17): 9547-57, 2013). As therapy for the infection and diarrheaassociated with GI.1 NoV, one strategy is a combination of the V_(H)Hantibodies 6.3 and 10.4, as they both illustrated the best overallblockade activity and they are directed toward different epitopes.Without being bound by theory, this is a characteristic that woulddiminish the probability of resistant mutant selection.

The GII.4 genotype is further classified into variants, when thedifference in the amino acid sequence of VP1 is more than 5%; with this5% of amino acid differences, a new variant is able to emerge into a newpandemy of NoV (Kroneman et al., Arch Virol 158(10): 2059-68, 2013).Several of the V_(H)H antibodies specific for GII.4 NoV not only showedcross-reactivity among the different variants of GII.4 tested from 1977to 2012, but also showed cross-reactivity among other genotypes withinthe GII genogroup thereby suggesting the future recognition of newvariants within the GII.4 genotype that could potentially generatefuture pandemic outbreaks (Lindesmith et al., J Virol 85(1): 231-42,2011; Lindesmith et al., PLoS Pathog 8(5): e1002705, 2012; Lindesmith etal., J Virol 86(2): 873-83, 2012; Lindesmith et al., J Virol 87(5):2803-13, 2013). The GII.4 V_(H)Hs clones 3.2, 4.1 and 5.4 all showedsimilar CDR3 amino acid sequences. These data, together with the resultsfrom the competition assays, suggest these antibodies are directedtoward the same epitope. Although they share the same CDR3, the V_(H)H3.2 displayed less cross reactivity with other GII genotypes differentfrom GII.4 compared to V_(H)H 5.4. This difference in specificity mayultimately be due to distinct amino acid changes in other regions of thesequences. The V_(H)H 1, 4.1, and 5.4 all showed the overall bestblockade profile, with the EC50 values ranging between 0.341 and 2.0μg/ml.

Example 7 Competition Assays

To evaluate the ability of a polyclonal serum to impair the V_(H)Hsbinding to NoV VLPs and vice versa, several competition assays wereconducted. When llama NoV strain specific hyperimmune serum was used asblockade antibodies, the binding of the V_(H)H 11.1 to the GI.1 NorwalkVLP was reduced 56%, while the binding of the other V_(H)Hs to the VLPswas reduced between 47% and 16% (FIG. 5). This result indicates thatV_(H)Hs were able to bind to their corresponding epitope within the Pdomain of the VLP even in the presence of high concentration ofconventional antibodies including those directed to the same epitopes.

The V_(H)Hs were able neither to block nor to compete with the bindingof the anti-NoV hyperimmune sera from llama to the VLPs, even when apool of all the V_(H)Hs was used, suggesting that they would not bedirected to immunodominant epitopes.

Given the results obtained in the surrogate neutralization assays, twoV_(H)H specific for each NoV genogroup were selected and labeled withAlexa fluor in order to elucidate if these clones were directed to thesame or a different epitope within the VP1 P domain. The competitionassays between the different V_(H)Hs indicated that the clones 6.3 and10.4 are directed to different epitopes and their binding sites are alsodifferent from the epitopes recognized by the other GI V_(H)Hs.Regarding the GII specific V_(H)Hs, clone 1 was directed to a distinctepitope to that recognized by V_(H)H 5.4 and the ones recognized by theother GII specific V_(H)Hs. The binding of labeled V_(H)H 5.4, wasimpaired by the presence of V_(H)H 3.2, V_(H)H 4.1 and V_(H)H 19according to the immunofluorescence assay results, suggesting that allthese clones are directed to the same epitope (FIG. 6). This observationis in agreement with the similarities detected in the CDR3 region of allthe nanoantibodies (FIG. 8).

Additionally, V_(H)Hs 6.3 and 104 are directed to differential epitopesto those recognized by conventional Mabs 2, 4, 7, 40 and 47 specific forGI.1 NoVs and V_(H)Hs 1 and 4.1 are directed to different epitopes tothose reacting with MAbs 3, 10 and 12, specific for GII.4 NoVs.

Example 8 Mapping of the Linear Epitope Recognized by V_(H)H 7.5

The V_(H)H 7.5 recognized all the GII strains tested by WB (FIG. 9) andELISA (Table 3) indicating that it was directed to a linear epitopewithin the P domain of VP1 according to the chimeric VLP ELISA results(Table 2).

To conduct the epitope mapping of this V_(H)H, a peptidic librarycorresponding to the P domain of the VP1 amino acid sequence from NoVToronto strain was synthesized. The library consisted of 67 peptidesthat were 17 amino acids long, with 5 amino acids overlapping with theprevious and the next peptide of the sequence. The V_(H)H 7.5 was testedagainst each of the peptides and a positive result was obtained for theoverlapping peptides corresponding to amino acids 512-528(GYFRFESWVNPFYTLAP, SEQ ID NO: 31) and 517-533 (ESWVNPFYTLAPMGTGN, SEQID NO: 32) while a negative result was obtained for the flankingoverlapping peptides 507-523 (TVPPNGYFRFESWVNPF, SEQ ID NO: 33) and522-538 (PFYTLAPMGTGNGRRRI, SEQ ID NO: 34) (FIG. 6). According to theseresults the sequences GYFRFESWVNPF (amino acids 1-12 of SEQ ID NO: 31)and PFYTLAPMGTGN (amino acids 6-17 of SEQ ID NO: 32) separately, werenot enough to be recognized by V_(H)H 7.5. Then, the putative epitopeshould be contained within the sequences of the two positive peptides,more specifically it should be comprised within the sequence from aminoacids 517 to 528 (ESWVNPFYTLAP, amino acids 1-12 of SEQ ID NO: 32). Thisregion of the P domain of VP1 protein is located close to the C-terminalend of the VP1 protein (FIG. 6b ).

The linear epitope of V_(H)H 7.5 was shown to be located at the Cterminal of NoV VP1, within the P1 subdomain, in concordance withprevious reports for cross reactive MAbs (Parker et al. J Virol 79(12):7402-9, 2005; Shiota et al. J Virol 81(22): 12298-306, 2007). Theepitope located within the protruding domain may be more accessible inthe native virion than epitopes located in the S domain, the target ofmost of other cross reactive MAbs (Parra et al., PLoS One 8(6): e67592,2013). Despite the lack of blockade activity of this V_(H)H, this V_(H)Hhas the potential for inhibition of Norovirus infection and diarrheathrough other mechanisms or to be useful in a diagnostic assay.

Example 9 Mapping of Conformational Putative Epitope of V_(H)H 16Binding

The V_(H)H 16 was specific for VP1 of the MD2004 strain and was not ableto recognize the other GII strains, including MD 145. For that reasonspecific mutants were designed targeting the differential amino acidsbetween both strains.

The immunofluorescence assay showed that the V_(H)H 16 failed torecognize a double mutant (G340A/E376Q) as well as a single mutant(G340A and E376Q) indicating that both amino acids 340 and 376 arecritical for the binding of this V_(H)H to VP1 and may be implicated inthe epitope, while mutations in the amino acids 294, 368 and 389 did notimpair V_(H)H 16 binding (FIG. 7a ). As a confirmation assay, VLPsrepresenting the double escape mutantG340A/E 376Q were developed,expressed and purified. Of the anti-MD2004 V_(H)H panel, only clone 16failed to recognize this mutant by ELISA (FIG. 7b ), in concordance withthe immunofluorescence assay.

Noroviruses (NoV) are recognized as a leading cause of viral food bornehuman gastroenteritis worldwide, affecting humans of all ages. It isconsidered to be the second leading cause of childhood diarrhea afterrotavirus. Additionally, NoV chronic infection represents a significantproblem in immunocompromised patients. The infectious oral dose isestimated to be less than 20 viral particles, with immunocompetentpatients usually suffering gastroenteritis followed by viral sheddingfor 20 to 40 days while immunocompromised patients usually becomechronically infected with viral shedding lasting weeks to years (BokandGreen. N Engl J Med 367(22): 2126-32, 2012; Green, Caliciviridae: TheNoroviruses. Fields Virology, 6th edition. H. P. Knipe D M, Griffin etal., Philadelphia: Lippincott Williams & Wilkins: 582-608, 2013).

Noroviruses present a broad genetic variability, with humans mainlyinfected with genogroups GI and GII. The latter is prevalent worldwidewith more than 30 genotypes being reported within this genogroup(Debbink, Lindesmith, et al., J Virol., Mar. 19, 2014; Kroneman, Vega,et al., Arch Virol 158(10): 2059-68, 2013). However, to date an in vitrocell culture system has not been develop to isolate and propagate thevirus, thus limiting antigenic studies. As such, the real antigenicdiversity, critical for a rational vaccine design, is still largelyunknown. Thus, vaccines or therapeutic interventions to prevent andtreat NoV diarrhea are therefore not yet available. Regarding vaccineeffective design, many questions remain, such as the identification ofthe proper antigens involved in virus neutralization and protection(Debbink et al., J Virol, Mar. 19, 2014; Debbink et al., J Virol, Apr.16, 2014).

Detailed knowledge of norovirus VP1 structure can be informed by thistype of analysis with nanobodies.

In view of the many possible embodiments to which the principles of ourinvention may be applied, it should be recognized that illustratedembodiments are only examples of the invention and should not beconsidered a limitation on the scope of the invention. Rather, the scopeof the invention is defined by the following claims. We therefore claimas our invention all that comes within the scope and spirit of theseclaims.

We claim:
 1. A method for detecting Norovirus in a sample, comprising:contacting the sample with an isolated V_(H)H monoclonal antibody or anantigen binding fragment thereof; and detecting the V_(H)H monoclonalantibody bound to the sample, wherein the presence of the V_(H)Hmonoclonal antibody bound to the sample indicates the presence of theNorovirus, wherein the V_(H)H monoclonal antibody specifically binds aNorovirus polypeptide and comprises a heavy chain variable domain,wherein the heavy chain variable domain comprises a heavy chaincomplementarity determining region (CDR)1, a CDR2 and a CDR3, andwherein: a) the CDR1 comprises amino acids 26-33 of SEQ ID NO: 3, theCDR2 comprises amino acids 51-57 of SEQ ID NO: 3, and the CDR3 comprisesamino acids 96-109 of SEQ ID NO: 3; b) the CDR1 comprises amino acids26-33 of SEQ ID NO: 2, the CDR2 comprises amino acids 51-57 of SEQ IDNO: 2, and the CDR3 comprises amino acids 96-109 of SEQ ID NO: 2; c) theCDR1 comprises amino acids 26-33 of SEQ ID NO: 1, the CDR2 comprisesamino acids 51-57 of SEQ ID NO: 1, and the CDR3 comprises amino acids96-109 of SEQ ID NO: 1; d) the CDR1 comprises amino acids 26-33 of SEQID NO: 4, the CDR2 comprises amino acids 51-57 of SEQ ID NO: 4, and theCDR3 comprises amino acids 96-109 of SEQ ID NO: 4; e) the CDR1 comprisesamino acids 26-33 of SEQ ID NO: 5, the CDR2 comprises amino acids 51-58of SEQ ID NO: 5, and the CDR3 comprises amino acids 97-110 of SEQ ID NO:5; f) the CDR1 comprises amino acids 26-33 of SEQ ID NO: 6, the CDR2comprises amino acids 51-58 of SEQ ID NO: 6, and the CDR3 comprisesamino acids 97-111 of SEQ ID NO: 6; g) the CDR1 comprises amino acids26-33 of SEQ ID NO: 7, the CDR2 comprises amino acids 51-58 of SEQ IDNO: 7, and the CDR3 comprises amino acids 97-111 of SEQ ID NO: 7; h) theCDR1 comprises amino acids 26-33 of SEQ ID NO: 8, the CDR2 comprisesamino acids 51-58 of SEQ ID NO: 8, and the CDR3 comprises amino acids97-111 of SEQ ID NO: 8; i) the CDR1 comprises amino acids 26-33 of SEQID NO: 9, the CDR2 comprises amino acids 51-57 of SEQ ID NO: 9, and theCDR3 comprises amino acids 96-112 of SEQ ID NO: 9; j) the CDR1 comprisesamino acids 26-33 of SEQ ID NO: 10, the CDR2 comprises amino acids 51-57of SEQ ID NO: 10, and the CDR3 comprises amino acids 96-112 of SEQ IDNO: 10; k) the CDR1 comprises amino acids 26-33 of SEQ ID NO: 11, theCDR2 comprises amino acids 51-57 of SEQ ID NO: 11, and the CDR3comprises amino acids 96-114 of SEQ ID NO: 11; l) the CDR1 comprisesamino acids 26-33 of SEQ ID NO: 12, the CDR2 comprises amino acids 51-57of SEQ ID NO: 12, and the CDR3 comprises amino acids 96-112 of SEQ IDNO: 12; m) the CDR1 comprises amino acids 26-33 of SEQ ID NO: 13, theCDR2 comprises amino acids 51-58 of SEQ ID NO: 13, and the CDR3comprises amino acids 97-113 of SEQ ID NO: 13; n) the CDR1 comprisesamino acids 26-33 of SEQ ID NO: 14, the CDR2 comprises amino acids 51-58of SEQ ID NO: 14, and the CDR3 comprises amino acids 97-113 of SEQ IDNO: 14; o) the CDR1 comprises amino acids 26-33 of SEQ ID NO: 15, theCDR2 comprises amino acids 51-58 of SEQ ID NO: 15, and the CDR3comprises amino acids 97-114 of SEQ ID NO: 15; p) the CDR1 comprisesamino acids 26-33 of SEQ ID NO: 16, the CDR2 comprises amino acids 51-57of SEQ ID NO: 16, and the CDR3 comprises amino acids 96-107 of SEQ IDNO: 16; q) the CDR1 comprises amino acids 26-33 of SEQ ID NO: 17, theCDR2 comprises amino acids 51-58 of SEQ ID NO: 17, and the CDR3comprises amino acids 97-113 of SEQ ID NO: 17; r) the CDR1 comprisesamino acids 26-33 of SEQ ID NO: 18, the CDR2 comprises amino acids 51-58of SEQ ID NO: 18, and the CDR3 comprises amino acids 97-114 of SEQ IDNO: 18; s) the CDR1 comprises amino acids 26-33 of SEQ ID NO: 19, theCDR2 comprises amino acids 51-58 of SEQ ID NO: 19, and the CDR3comprises amino acids 97-113 of SEQ ID NO: 19; t) the CDR1 comprisesamino acids 26-33 of SEQ ID NO: 20, the CDR2 comprises amino acids 51-57of SEQ ID NO:20, and the CDR3 comprises amino acids 96-111 of SEQ ID NO:20; u) the CDR1 comprises amino acids 26-33 of SEQ ID NO: 21, the CDR2comprises amino acids 51-57 of SEQ ID NO:21, and the CDR3 comprisesamino acids 96-102 of SEQ ID NO: 21; v) the CDR1 comprises amino acids26-33 of SEQ ID NO: 22, the CDR2 comprises amino acids 51-57 of SEQ IDNO:22, and the CDR3 comprises amino acids 96-110 of SEQ ID NO: 22; w)the CDR1 comprises amino acids 26-33 of SEQ ID NO: 23, the CDR2comprises amino acids 50-56 of SEQ ID NO:23, and the CDR3 comprisesamino acids 95-104 of SEQ ID NO: 23; x) the CDR1 comprises amino acids26-33 of SEQ ID NO: 24, the CDR2 comprises amino acids 51-57 of SEQ IDNO:24, and the CDR3 comprises amino acids 96-107 of SEQ ID NO: 24; y)the CDR1 comprises amino acids 26-33 of SEQ ID NO: 25, the CDR2comprises amino acids 51-57 of SEQ ID NO: 25, and the CDR3 comprisesamino acids 100-110 of SEQ ID NO: 25; z) the CDR1 comprises amino acids26-33 of SEQ ID NO: 26, the CDR2 comprises amino acids 50-57 of SEQ IDNO:26, and the CDR3 comprises amino acids 96-117 of SEQ ID NO: 26; aa)the CDR1 comprises amino acids 26-33 of SEQ ID NO: 27, the CDR2comprises amino acids 51-58 of SEQ ID NO:27, and the CDR3 comprisesamino acids 97-108 of SEQ ID NO: 27; bb) the CDR1 comprises amino acids26-33 of SEQ ID NO: 28, the CDR2 comprises amino acids 51-57 of SEQ IDNO:28, and the CDR3 comprises amino acids 96-112 of SEQ ID NO: 28; cc)the CDR1 comprises amino acids 26-33 of SEQ ID NO: 29, the CDR2comprises amino acids 51-58 of SEQ ID NO:29, and the CDR3 comprisesamino acids 97-115 of SEQ ID NO: 29; or dd) the CDR1 comprises aminoacids 26-33 of SEQ ID NO: 30, the CDR2 comprises amino acids 51-58 ofSEQ ID NO:30, and the CDR3 comprises amino acids 97-121 of SEQ ID NO:30, thereby detecting the Norovirus in the sample.
 2. The method ofclaim 1, wherein the V_(H)H monoclonal antibody or an antigen bindingfragment thereof is directly labeled.
 3. The method of claim 1, furthercomprising: contacting the sample with a second antibody thatspecifically binds the V_(H)H monoclonal antibody or an antigen bindingfragment thereof; and detecting the binding of the second antibody,wherein an increase in binding of the second antibody to the sample ascompared to binding of the second antibody to a control sample detectsthe presence of the Norovirus in the sample.
 4. The method of claim 1,wherein the Norovirus is Genogroup I Norovirus.
 5. The method of claim1, wherein the Norovirus is a Genogroup II Norovirus.
 6. The method ofclaim 1, wherein: a) the CDR1 comprises amino acids 26-33 of SEQ ID NO:3, the CDR2 comprises amino acids 51-57 of SEQ ID NO: 3, and the CDR3comprises amino acids 96-109 of SEQ ID NO: 3, and wherein the heavychain variable domain of the V_(H)H monoclonal antibody is at least 90%identical to SEQ ID NO: 3; b) the CDR1 comprises amino acids 26-33 ofSEQ ID NO: 2, the CDR2 comprises amino acids 51-57 of SEQ ID NO: 2, andthe CDR3 comprises amino acids 96-109 of SEQ ID NO: 2, and wherein theheavy chain variable domain of the V_(H)H monoclonal antibody is atleast 90% identical to SEQ ID NO: 2; c) the CDR1 comprises amino acids26-33 of SEQ ID NO: 1, the CDR2 comprises amino acids 51-57 of SEQ IDNO: 1, and the CDR3 comprises amino acids 96-109 of SEQ ID NO: 1, andwherein the heavy chain variable domain of the V_(H)H monoclonalantibody is at least 90% identical to SEQ ID NO: 1; d) the CDR1comprises amino acids 26-33 of SEQ ID NO: 4, the CDR2 comprises aminoacids 51-57 of SEQ ID NO: 4, and the CDR3 comprises amino acids 96-109of SEQ ID NO: 4, and wherein the heavy chain variable domain of theV_(H)H monoclonal antibody is at least 90% identical to SEQ ID NO: 4; e)the CDR1 comprises amino acids 26-33 of SEQ ID NO: 5, the CDR2 comprisesamino acids 51-58 of SEQ ID NO: 5, and the CDR3 comprises amino acids97-110 of SEQ ID NO: 5, and wherein the heavy chain variable domain ofthe V_(H)H monoclonal antibody is at least 90% identical to SEQ ID NO:5; f) the CDR1 comprises amino acids 26-33 of SEQ ID NO: 6, the CDR2comprises amino acids 51-58 of SEQ ID NO: 6, and the CDR5 comprisesamino acids 97-111 of SEQ ID NO: 6, and wherein the heavy chain variabledomain of the V_(H)H monoclonal antibody is at least 90% identical toSEQ ID NO: 6; g) the CDR1 comprises amino acids 26-33 of SEQ ID NO: 7,the CDR2 comprises amino acids 51-58 of SEQ ID NO: 7, and the CDR3comprises amino acids 97-111 of SEQ ID NO: 7, and wherein the heavychain variable domain of the V_(H)H monoclonal antibody is at least 90%identical to SEQ ID NO: 7; h) the CDR1 comprises amino acids 26-33 ofSEQ ID NO: 8, the CDR2 comprises amino acids 51-58 of SEQ ID NO: 8, andthe CDR3 comprises amino acids 97-111 of SEQ ID NO: 8, and wherein theheavy chain variable domain of the V_(H)H monoclonal antibody is atleast 90% identical to SEQ ID NO: 8; i) the CDR1 comprises amino acids26-33 of SEQ ID NO: 9, the CDR2 comprises amino acids 51-57 of SEQ IDNO: 9, and the CDR3 comprises amino acids 96-112 of SEQ ID NO: 9, andwherein the heavy chain variable domain of the V_(H)H monoclonalantibody is at least 90% identical to SEQ ID NO: 9; j) the CDR1comprises amino acids 26-33 of SEQ ID NO: 10, the CDR2 comprises aminoacids 51-57 of SEQ ID NO: 10, and the CDR3 comprises amino acids 96-112of SEQ ID NO: 10, and wherein the heavy chain variable domain of theV_(H)H monoclonal antibody is at least 90% identical to SEQ ID NO: 10;k) the CDR1 comprises amino acids 26-33 of SEQ ID NO: 11, the CDR2comprises amino acids 51-57 of SEQ ID NO: 11, and the CDR3 comprisesamino acids 96-114 of SEQ ID NO: 11, and wherein the heavy chainvariable domain of the V_(H)H monoclonal antibody is at least 90%identical to SEQ ID NO: 11; l) the CDR1 comprises amino acids 26-33 ofSEQ ID NO: 12, the CDR2 comprises amino acids 51-57 of SEQ ID NO: 12,and the CDR3 comprises amino acids 96-112 of SEQ ID NO: 12, and whereinthe heavy chain variable domain of the V_(H)H monoclonal antibody is atleast 90% identical to SEQ ID NO: 12; m) the CDR1 comprises amino acids26-33 of SEQ ID NO: 13, the CDR2 comprises amino acids 51-58 of SEQ IDNO: 13, and the CDR3 comprises amino acids 97-113 of SEQ ID NO: 13, andwherein the heavy chain variable domain of the V_(H)H monoclonalantibody is at least 90% identical to SEQ ID NO: 13; n) the CDR1comprises amino acids 26-33 of SEQ ID NO: 14, the CDR2 comprises aminoacids 51-58 of SEQ ID NO: 14, and the CDR3 comprises amino acids 97-113of SEQ ID NO: 14, and wherein the heavy chain variable domain of theV_(H)H monoclonal antibody is at least 90% identical to SEQ ID NO: 14;o) the CDR1 comprises amino acids 26-33 of SEQ ID NO: 15, the CDR2comprises amino acids 51-58 of SEQ ID NO: 15, and the CDR3 comprisesamino acids 97-114 of SEQ ID NO: 15, and wherein the heavy chainvariable domain of the V_(H)H monoclonal antibody is at least 90%identical to SEQ ID NO: 15; p) the CDR1 comprises amino acids 26-33 ofSEQ ID NO: 16, the CDR2 comprises amino acids 51-57 of SEQ ID NO: 16,and the CDR3 comprises amino acids 96-107 of SEQ ID NO: 16, and whereinthe heavy chain variable domain of the V_(H)H monoclonal antibody is atleast 90% identical to SEQ ID NO: 16; q) the CDR1 comprises amino acids26-33 of SEQ ID NO: 17, the CDR2 comprises amino acids 51-58 of SEQ IDNO: 17, and the CDR3 comprises amino acids 97-113 of SEQ ID NO: 17, andwherein the heavy chain variable domain of the V_(H)H monoclonalantibody is at least 90% identical to SEQ ID NO: 17; r) the CDR1comprises amino acids 26-33 of SEQ ID NO: 18, the CDR2 comprises aminoacids 51-58 of SEQ ID NO: 18, and the CDR3 comprises amino acids 97-114of SEQ ID NO: 18, and wherein the heavy chain variable domain of theV_(H)H monoclonal antibody is at least 90% identical to SEQ ID NO: 18;s) the CDR1 comprises amino acids 26-33 of SEQ ID NO: 19, the CDR2comprises amino acids 51-58 of SEQ ID NO: 19, and the CDR3 comprisesamino acids 97-113 of SEQ ID NO: 19, and wherein the heavy chainvariable domain of the V_(H)H monoclonal antibody is at least 90%identical to SEQ ID NO: 19; t) the CDR1 comprises amino acids 26-33 ofSEQ ID NO: 20, the CDR2 comprises amino acids 51-57 of SEQ ID NO:20, andthe CDR3 comprises amino acids 96-111 of SEQ ID NO: 20, and wherein theheavy chain variable domain of the V_(H)H monoclonal antibody is atleast 90% identical to SEQ ID NO: 20; or u) the CDR1 comprises aminoacids 26-33 of SEQ ID NO: 21, the CDR2 comprises amino acids 51-57 ofSEQ ID NO:21, and the CDR3 comprises amino acids 96-102 of SEQ ID NO:21, and wherein the heavy chain variable domain of the V_(H)H monoclonalantibody is at least 90% identical to SEQ ID NO:
 21. 7. The method ofclaim 1, wherein: a) the CDR1 comprises amino acids 26-33 of SEQ ID NO:22, the CDR2 comprises amino acids 51-57 of SEQ ID NO:22, and the CDR3comprises amino acids 96-110 of SEQ ID NO: 22, and wherein the heavychain variable domain of the V_(H)H monoclonal antibody is at least 90%identical to SEQ ID NO: 22; b) the CDR1 comprises amino acids 26-33 ofSEQ ID NO: 23, the CDR2 comprises amino acids 50-56 of SEQ ID NO:23, andthe CDR3 comprises amino acids 95-104 of SEQ ID NO: 23, and wherein theheavy chain variable domain of the V_(H)H monoclonal antibody is atleast 90% identical to SEQ ID NO: 23; c) the CDR1 comprises amino acids26-33 of SEQ ID NO: 24, the CDR2 comprises amino acids 51-57 of SEQ IDNO:24, and the CDR3 comprises amino acids 96-107 of SEQ ID NO: 24, andwherein the heavy chain variable domain of the V_(H)H monoclonalantibody is at least 90% identical to SEQ ID NO: 24; d) the CDR1comprises amino acids 26-33 of SEQ ID NO: 25, the CDR2 comprises aminoacids 51-57 of SEQ ID NO: 25, and the CDR3 comprises amino acids 100-110of SEQ ID NO: 25, and wherein the heavy chain variable domain of theV_(H)H monoclonal antibody is at least 90% identical to SEQ ID NO: 25;e) the CDR1 comprises amino acids 26-33 of SEQ ID NO: 26, the CDR2comprises amino acids 50-57 of SEQ ID NO:26, and the CDR3 comprisesamino acids 96-117 of SEQ ID NO: 26, and wherein the heavy chainvariable domain of the V_(H)H monoclonal antibody is at least 90%identical to SEQ ID NO: 26; f) the CDR1 comprises amino acids 26-33 ofSEQ ID NO: 27, the CDR2 comprises amino acids 51-58 of SEQ ID NO:27, andthe CDR3 comprises amino acids 97-108 of SEQ ID NO: 27, and wherein theheavy chain variable domain of the V_(H)H monoclonal antibody is atleast 90% identical to SEQ ID NO: 27; g) the CDR1 comprises amino acids26-33 of SEQ ID NO: 28, the CDR2 comprises amino acids 51-57 of SEQ IDNO:28, and the CDR3 comprises amino acids 96-112 of SEQ ID NO: 28, andwherein the heavy chain variable domain of the V_(H)H monoclonalantibody is at least 90% identical to SEQ ID NO: 28; h) the CDR1comprises amino acids 26-33 of SEQ ID NO: 29, the CDR2 comprises aminoacids 51-58 of SEQ ID NO:29, and the CDR3 comprises amino acids 97-115of SEQ ID NO: 29, and wherein the heavy chain variable domain of theV_(H)H monoclonal antibody is at least 90% identical to SEQ ID NO: 29;or i) the CDR1 comprises amino acids 26-33 of SEQ ID NO: 30, the CDR2comprises amino acids 51-58 of SEQ ID NO:30, and the CDR3 comprisesamino acids 97-121 of SEQ ID NO: 30, and wherein the heavy chainvariable domain of the V_(H)H monoclonal antibody is at least 90%identical to SEQ ID NO:
 30. 8. The method of claim 6, wherein the heavychain variable domain comprises the amino acid sequence set for the asone of SEQ ID NOs: 1-21.
 9. The method of claim 7, wherein the heavychain variable domain comprises the amino acid sequence set forth as oneof SEQ ID NOs: 22-30.
 10. The method of claim 1, wherein the sample is atissue, blood, serum, plasma, spinal fluid, sputum, nasopharyngealsecretion, stool or urine sample.
 11. The method of claim 1, wherein thesample is an environmental sample.
 12. The method of claim 1, whereinthe sample comprise a subunit vaccine.
 13. The method of claim 2,wherein the label is an enzyme, prosthetic group, fluorescent material,luminescent material, magnetic agent or radioactive material.
 14. A kitfor detecting a Norovirus, comprising: i) a container comprising anisolated V_(H)H monoclonal antibody or an antigen binding fragmentthereof, wherein the V_(H)H monoclonal antibody specifically binds aNorovirus polypeptide and comprises a heavy chain variable domain,wherein the heavy chain variable domain comprises a heavy chaincomplementarity determining region (CDR)1, a CDR2 and a CDR3, andwherein: a) the CDR1 comprises amino acids 26-33 of SEQ ID NO: 3, theCDR2 comprises amino acids 51-57 of SEQ ID NO: 3, and the CDR3 comprisesamino acids 96-109 of SEQ ID NO: 3; b) the CDR1 comprises amino acids26-33 of SEQ ID NO: 2, the CDR2 comprises amino acids 51-57 of SEQ IDNO: 2, and the CDR3 comprises amino acids 96-109 of SEQ ID NO: 2; c) theCDR1 comprises amino acids 26-33 of SEQ ID NO: 1, the CDR2 comprisesamino acids 51-57 of SEQ ID NO: 1, and the CDR3 comprises amino acids96-109 of SEQ ID NO: 1; d) the CDR1 comprises amino acids 26-33 of SEQID NO: 4, the CDR2 comprises amino acids 51-57 of SEQ ID NO: 4, and theCDR3 comprises amino acids 96-109 of SEQ ID NO: 4; e) the CDR1 comprisesamino acids 26-33 of SEQ ID NO: 5, the CDR2 comprises amino acids 51-58of SEQ ID NO: 5, and the CDR3 comprises amino acids 97-110 of SEQ ID NO:5; f) the CDR1 comprises amino acids 26-33 of SEQ ID NO: 6, the CDR2comprises amino acids 51-58 of SEQ ID NO: 6, and the CDR3 comprisesamino acids 97-111 of SEQ ID NO: 6; g) the CDR1 comprises amino acids26-33 of SEQ ID NO: 7, the CDR2 comprises amino acids 51-58 of SEQ IDNO: 7, and the CDR3 comprises amino acids 97-111 of SEQ ID NO: 7; h) theCDR1 comprises amino acids 26-33 of SEQ ID NO: 8, the CDR2 comprisesamino acids 51-58 of SEQ ID NO: 8, and the CDR3 comprises amino acids97-111 of SEQ ID NO: 8; i) the CDR1 comprises amino acids 26-33 of SEQID NO: 9, the CDR2 comprises amino acids 51-57 of SEQ ID NO: 9, and theCDR3 comprises amino acids 96-112 of SEQ ID NO: 9; j) the CDR1 comprisesamino acids 26-33 of SEQ ID NO: 10, the CDR2 comprises amino acids 51-57of SEQ ID NO: 10, and the CDR3 comprises amino acids 96-112 of SEQ IDNO: 10; k) the CDR1 comprises amino acids 26-33 of SEQ ID NO: 11, theCDR2 comprises amino acids 51-57 of SEQ ID NO: 11, and the CDR3comprises amino acids 96-114 of SEQ ID NO: 11; l) the CDR1 comprisesamino acids 26-33 of SEQ ID NO: 12, the CDR2 comprises amino acids 51-57of SEQ ID NO: 12, and the CDR3 comprises amino acids 96-112 of SEQ IDNO: 12; m) the CDR1 comprises amino acids 26-33 of SEQ ID NO: 13, theCDR2 comprises amino acids 51-58 of SEQ ID NO: 13, and the CDR3comprises amino acids 97-113 of SEQ ID NO: 13; n) the CDR1 comprisesamino acids 26-33 of SEQ ID NO: 14, the CDR2 comprises amino acids 51-58of SEQ ID NO: 14, and the CDR3 comprises amino acids 97-113 of SEQ IDNO: 14; o) the CDR1 comprises amino acids 26-33 of SEQ ID NO: 15, theCDR2 comprises amino acids 51-58 of SEQ ID NO: 15, and the CDR3comprises amino acids 97-114 of SEQ ID NO: 15; p) the CDR1 comprisesamino acids 26-33 of SEQ ID NO: 16, the CDR2 comprises amino acids 51-57of SEQ ID NO: 16, and the CDR3 comprises amino acids 96-107 of SEQ IDNO: 16; q) the CDR1 comprises amino acids 26-33 of SEQ ID NO: 17, theCDR2 comprises amino acids 51-58 of SEQ ID NO: 17, and the CDR3comprises amino acids 97-113 of SEQ ID NO: 17; r) the CDR1 comprisesamino acids 26-33 of SEQ ID NO: 18, the CDR2 comprises amino acids 51-58of SEQ ID NO: 18, and the CDR3 comprises amino acids 97-114 of SEQ IDNO: 18; s) the CDR1 comprises amino acids 26-33 of SEQ ID NO: 19, theCDR2 comprises amino acids 51-58 of SEQ ID NO: 19, and the CDR3comprises amino acids 97-113 of SEQ ID NO: 19; t) the CDR1 comprisesamino acids 26-33 of SEQ ID NO: 20, the CDR2 comprises amino acids 51-57of SEQ ID NO:20, and the CDR3 comprises amino acids 96-111 of SEQ ID NO:20; u) the CDR1 comprises amino acids 26-33 of SEQ ID NO: 21, the CDR2comprises amino acids 51-57 of SEQ ID NO:21, and the CDR3 comprisesamino acids 96-102 of SEQ ID NO: 21; v) the CDR1 comprises amino acids26-33 of SEQ ID NO: 22, the CDR2 comprises amino acids 51-57 of SEQ IDNO:22, and the CDR3 comprises amino acids 96-110 of SEQ ID NO: 22; w)the CDR1 comprises amino acids 26-33 of SEQ ID NO: 23, the CDR2comprises amino acids 50-56 of SEQ ID NO:23, and the CDR3 comprisesamino acids 95-104 of SEQ ID NO: 23; x) the CDR1 comprises amino acids26-33 of SEQ ID NO: 24, the CDR2 comprises amino acids 51-57 of SEQ IDNO:24, and the CDR3 comprises amino acids 96-107 of SEQ ID NO: 24; y)the CDR1 comprises amino acids 26-33 of SEQ ID NO: 25, the CDR2comprises amino acids 51-57 of SEQ ID NO: 25, and the CDR3 comprisesamino acids 100-110 of SEQ ID NO: 25; z) the CDR1 comprises amino acids26-33 of SEQ ID NO: 26, the CDR2 comprises amino acids 50-57 of SEQ IDNO:26, and the CDR3 comprises amino acids 96-117 of SEQ ID NO: 26; aa)the CDR1 comprises amino acids 26-33 of SEQ ID NO: 27, the CDR2comprises amino acids 51-58 of SEQ ID NO:27, and the CDR3 comprisesamino acids 97-108 of SEQ ID NO: 27; bb) the CDR1 comprises amino acids26-33 of SEQ ID NO: 28, the CDR2 comprises amino acids 51-57 of SEQ IDNO:28, and the CDR3 comprises amino acids 96-112 of SEQ ID NO: 28; cc)the CDR1 comprises amino acids 26-33 of SEQ ID NO: 29, the CDR2comprises amino acids 51-58 of SEQ ID NO:29, and the CDR3 comprisesamino acids 97-115 of SEQ ID NO: 29; or dd) the CDR1 comprises aminoacids 26-33 of SEQ ID NO: 30, the CDR2 comprises amino acids 51-58 ofSEQ ID NO:30, and the CDR3 comprises amino acids 97-121 of SEQ ID NO:30; and ii) instructions for using the kit.
 15. The kit of claim 14,wherein the kit further comprises: iii) a container comprising a secondmonoclonal antibody that specifically binds the V_(H)H monoclonalantibody or antigen binding fragment thereof.
 16. The kit of claim 14,wherein the V_(H)H monoclonal antibody or antigen binding fragment islabeled.
 17. The kit of claim 14, further comprising a containingcomprising a reagent or a buffer.
 18. A composition comprising, atherapeutically effective amount of an isolated V_(H)H monoclonalantibody or an antigen binding fragment thereof, wherein the isolatedV_(H)H monoclonal antibody specifically binds a Norovirus polypeptideand comprises a heavy chain variable domain, wherein the heavy chainvariable domain comprises a heavy chain complementarity determiningregion (CDR)1, a CDR2 and a CDR3, and wherein: a) the CDR1 comprisesamino acids 26-33 of SEQ ID NO: 3, the CDR2 comprises amino acids 51-57of SEQ ID NO: 3, and the CDR3 comprises amino acids 96-109 of SEQ ID NO:3; b) the CDR1 comprises amino acids 26-33 of SEQ ID NO: 2, the CDR2comprises amino acids 51-57 of SEQ ID NO: 2, and the CDR3 comprisesamino acids 96-109 of SEQ ID NO: 2; c) the CDR1 comprises amino acids26-33 of SEQ ID NO: 1, the CDR2 comprises amino acids 51-57 of SEQ IDNO: 1, and the CDR3 comprises amino acids 96-109 of SEQ ID NO: 1; d) theCDR1 comprises amino acids 26-33 of SEQ ID NO: 4, the CDR2 comprisesamino acids 51-57 of SEQ ID NO: 4, and the a CDR3 comprises amino acids96-109 of SEQ ID NO: 4; e) the CDR1 comprises amino acids 26-33 of SEQID NO: 5, the CDR2 comprises amino acids 51-58 of SEQ ID NO: 5, and theCDR3 comprises amino acids 97-110 of SEQ ID NO: 5; f) the CDR1 comprisesamino acids 26-33 of SEQ ID NO: 6, the CDR2 comprises amino acids 51-58of SEQ ID NO: 6, and the CDR3 comprises amino acids 97-111 of SEQ ID NO:6; g) the CDR1 comprises amino acids 26-33 of SEQ ID NO: 7, the CDR2comprises amino acids 51-58 of SEQ ID NO: 7, and the CDR3 comprisesamino acids 97-111 of SEQ ID NO: 7; h) the CDR1 comprises amino acids26-33 of SEQ ID NO: 8, the CDR2 comprises amino acids 51-58 of SEQ IDNO: 8, and the CDR3 comprises amino acids 97-111 of SEQ ID NO: 8; i) theCDR1 comprises amino acids 26-33 of SEQ ID NO: 9, the CDR2 comprisesamino acids 51-57 of SEQ ID NO: 9, and the CDR3 comprises amino acids96-112 of SEQ ID NO: 9; j) the CDR1 comprises amino acids 26-33 of SEQID NO: 10, the CDR2 comprises amino acids 51-57 of SEQ ID NO: 10, andthe CDR3 comprises amino acids 96-112 of SEQ ID NO: 10; k) the CDR1comprises amino acids 26-33 of SEQ ID NO: 11, the CDR2 comprises aminoacids 51-57 of SEQ ID NO: 11, and the CDR3 comprises amino acids 96-114of SEQ ID NO: 11; l) the CDR1 comprises amino acids 26-33 of SEQ ID NO:12, the CDR2 comprises amino acids 51-57 of SEQ ID NO: 12, and the CDR3comprises amino acids 96-112 of SEQ ID NO: 12; m) the CDR1 comprisesamino acids 26-33 of SEQ ID NO: 13, the CDR2 comprises amino acids 51-58of SEQ ID NO: 13, and the CDR3 comprises amino acids 97-113 of SEQ IDNO: 13; n) the CDR1 comprises amino acids 26-33 of SEQ ID NO: 14, theCDR2 comprises amino acids 51-58 of SEQ ID NO: 14, and the CDR3comprises amino acids 97-113 of SEQ ID NO: 14; o) the CDR1 comprisesamino acids 26-33 of SEQ ID NO: 15, the CDR2 comprises amino acids 51-58of SEQ ID NO: 15, and the CDR3 comprises amino acids 97-114 of SEQ IDNO: 15; p) the CDR1 comprises amino acids 26-33 of SEQ ID NO: 16, theCDR2 comprises amino acids 51-57 of SEQ ID NO: 16, and the CDR3comprises amino acids 96-107 of SEQ ID NO: 16; q) the CDR1 comprisesamino acids 26-33 of SEQ ID NO: 17, the CDR2 comprises amino acids 51-58of SEQ ID NO: 17, and the CDR3 comprises amino acids 97-113 of SEQ IDNO: 17; r) the CDR1 comprises amino acids 26-33 of SEQ ID NO: 18, theCDR2 comprises amino acids 51-58 of SEQ ID NO: 18, and the CDR3comprises amino acids 97-114 of SEQ ID NO: 18; s) the CDR1 comprisesamino acids 26-33 of SEQ ID NO: 19, the CDR2 comprises amino acids 51-58of SEQ ID NO: 19, and the CDR3 comprises amino acids 97-113 of SEQ IDNO: 19; t) the CDR1 comprises amino acids 26-33 of SEQ ID NO: 20, theCDR2 comprises amino acids 51-57 of SEQ ID NO:20, and the CDR3 comprisesamino acids 96-111 of SEQ ID NO: 20; u) the CDR1 comprises amino acids26-33 of SEQ ID NO: 21, the CDR2 comprises amino acids 51-57 of SEQ IDNO:21, and the CDR3 comprises amino acids 96-102 of SEQ ID NO: 21; v)the CDR1 comprises amino acids 26-33 of SEQ ID NO: 22, the CDR2comprises amino acids 51-57 of SEQ ID NO:22, and the CDR3 comprisesamino acids 96-110 of SEQ ID NO: 22; w) the CDR1 comprises amino acids26-33 of SEQ ID NO: 23, the CDR2 comprises amino acids 50-56 of SEQ IDNO:23, and the CDR3 comprises amino acids 95-104 of SEQ ID NO: 23; x)the CDR1 comprises amino acids 26-33 of SEQ ID NO: 24, the CDR2comprises amino acids 51-57 of SEQ ID NO:24, and the CDR3 comprisesamino acids 96-107 of SEQ ID NO: 24; y) the CDR1 comprises amino acids26-33 of SEQ ID NO: 25, the CDR2 comprises amino acids 51-57 of SEQ IDNO: 25, and the CDR3 comprises amino acids 100-110 of SEQ ID NO: 25; z)the CDR1 comprises amino acids 26-33 of SEQ ID NO: 26, the CDR2comprises acids 50-57 of SEQ ID NO:26, and the CDR3 comprises aminoacids 96-117 of SEQ ID NO: 26; aa) the CDR1 comprises amino acids 26-33of SEQ ID NO: 27, the CDR2 comprises acids 51-58 of SEQ ID NO:27, andthe CDR3 comprises amino acids 97-108 of SEQ ID NO: 27; bb) the CDR1comprises amino acids 26-33 of SEQ ID NO: 28, the CDR2 comprises acids51-57 of SEQ ID NO:28, and the CDR3 comprises amino acids 96-112 of SEQID NO: 28; cc) the CDR1 comprises amino acids 26-33 of SEQ ID NO: 29,the CDR2 comprises acids 51-58 of SEQ ID NO:29, and the CDR3 comprisesamino acids 97-115 of SEQ ID NO: 29; or dd) the CDR1 comprises aminoacids 26-33 of SEQ ID NO: 30, the CDR2 comprises acids 51-58 of SEQ IDNO:30, and the CDR3 comprises amino acids 97-121 of SEQ ID NO: 30,wherein the composition is formulated for enteric administration. 19.The composition of claim 18, further comprising an anti-viral agent. 20.A method of treating a subject with a Norovirus infection, comprisingadministering to the subject a therapeutically effective amount of thecomposition of claim 18, thereby treating the Norovirus infection in thesubject.
 21. The method of claim 20, wherein the subject is treated withmultiple doses of the composition.
 22. The method of claim 21, whereinthe subject is treated for about five to about ten days.
 23. The methodof claim 21, wherein the subject is immunocompromised.
 24. The method ofclaim 21, wherein the subject is a human child.